ФИЗИОЛОГИЯ РАСТЕНИЙ, 2015, том 62, № 5, с. 745-750
TOTAL PHENOL CONTENT AND TOTAL ANTIOXIDANT ACTIVITY DROP DURING Tacitus bellus DIRECT SHOOT ORGANOGENESIS1 © 2015 A. MitroviC, V. Maksimovic, D. Mutavd zic, J. Bogdanovic Pristov
Institute for Multidisciplinary Research, University of Belgrade, Belgrade, Serbia Received September 15, 2014
Changes in total phenolics content (TPC) and total antioxidant activity (TAA) in different morphogenic stages of Tacitus bellus (L.) Moran and J. Meyran (Crassulaceae) direct shoot organogenesis in vitro were examined. A HPLC method was used for the assessment of changes in phenolic profile. Significant decrease in TPC and TAA, as well as the decline of the amount of specific phenolic compounds coincided with the initiation of shoot organogenesis. Additional decrease in TPC and TAA, and the disappearance of procyanidin glycoside, occurred associated with shoot development. Performed correlation analysis suggests phenolics as the main component of nonenzymatic antioxidant system involved in T. bellus direct shoot organogenesis, it argues in favor of involvement of specific phenolics in the regulation of early stages of T. bellus direct shoot organogenesis and indicates different fine regulatory mechanisams of early and late stages of shoot organogenesis.
Keywords: Tacitus bellus — phenolics — shoot organogenesis — total antioxidant activity
De novo shoot and root organogenesis relies on totipotency of special populations of cells, that can be considered adult stem cells, characterized by capacity to regenerate in vitro the entire plant . The aux-in/cytokinin ratio in the medium is of the key importance for de novo organogenesis from plant tissues. It varies between plant tissues, genotypes, species, and even different explants of the same plant species . High cytokinin to auxin ratio can trigger the initiation of shoot meristems from undifferentiated callus . In the shoot meristem, cytokinin promotes stem-cell proliferation and inhibits stem-cell differentiation, while auxin triggers organ primordium initiation through repressing cytokinin biosynthesis .
Phenolics are diverse secondary metabolites abundant in plant tissues with many different physiological roles. The phenolic pool varies in composition as well as in amount between organisms, tissues, developmental stages, and in relation to environmental conditions . They are involved in growth regulation, organogenesis, dormancy control and in plant defense reac-
1 This text was submitted by the authors in English.
Abbreviations: BAP — benzylaminopurine; CAT — catalase; SOD — superoxide dismutase; POD — peroxidase; PPO — polyphenol oxidase; NAA — naphtaleneacetic acid; TAA — total antioxidant activity; TPC — total phenolics content.
Corresponding author: Aleksandra Mitrovic. Institute for Multidisciplinary Research, University of Belgrade, Kneza Viseslava 1, Belgrade, Serbia; fax: (+381) 113055289; e-mail: email@example.com
tion. Phenolics are oxidized by peroxidases (PODs) and polyphenol oxidase (PPO), and they act in reactive oxygen species (ROS) scavenging. It was shown that phenolics might be related to processes that control organogenesis through controlling auxin/cytokinin balance . Plant phenolics are modulators of IAA ca-tabolism. Arnaldos et al.  reported that some monophenols like synapic and ferulic acids, at low concentrations, inhibit enzymatic oxidation of IAA, while Jacobs and Rubery  presented that some fla-vonoids at physiological concentrations inhibit (quer-cetin) or stimulate (kaempferol) peroxidase catalyzed oxidation of IAA. Flavonoid accumulation in regions of high auxin concentration may reflect its protective mechanism from ROS generation during IAA catabo-lism or conjugation to inactive forms [9, 10].
The evidences for potential role of phenolic compounds in de novo organogenesis accumulated in last few decades. In general, many findings suggest inverse correlation between accumulation of phenolic substances and emergence of organized structures in organogenic cultures: strawberry , Momordica charantia , Hypericum mysorense , Sterculia urens .
The total antioxidant activity (TAA) comprises the contribution of nonenzymatic constituents with anti-oxidant capacity (ascorbate, glutathione, phenolics and sugars).
In our previous work, changes in the activities of antioxidative enzymes and isoforms during Tacitus bellus direct shoot organogenesis  were discussed. In present study, in order to elucidate the contribution
of phenolic substances in T. bellus shoot organogenesis, changes in TPC and TAA were examined and correlated with previously presented  changes in the activities of enzymes involved in phenolic metabolism (peroxidases and polyphenol oxidases). Furthermore, assessment of changes in phenolic profile during T. bellus organogenesis has been made.
MATERIALS AND METHODS
Plant material. The experiments were carried out on a small frost sensitive succulent from Mexico and widely cultivated horticultural subject, Tacitus bellus Moran and J. Meyran, syn. Graptopetalum bellum, Crassulace-ae. Direct shoot organogenesis was obtained on MS  medium supplemented with sucrose (30 g/L), ben-zylaminopurine (BAP, 0.1 mg/L) and naphtaleneacetic acid (NAA, 0.1 mg/L) and agar (0.7%) during 60 days . Primary explants were lower rosette leaves from in vitro multiplied and on hormone free medium rooted plants. Temperature in growth chambers was 25 ± 2°C. Four samples (5 explants per sample) were collected at 0, 10, 20, 40, and 60 days of culturing, frozen, powdered in the liquid nitrogen in a mortar, and used for analysis. The experiment was repeated twice.
ABTS-test. The total antioxidant activity was measured using ABTS/HRP end-point method, according to Arnao et al. . The reaction mixture contained 2 mM ABTS, 15 |M hydrogen peroxide, 0.25 |M HRP and 20 |L of 80% methanol extract of the powdered samples in 50 mM phosphate buffer, pH 7.5, in a total volume 2 mL. The assay temperature was 25°C. The reaction was monitored at 730 nm using a Shimadzu UV-160 spectrophotometer until a stable absorbance, due to ABTS radical formation, was obtained. Afterwards, different concentrations of ascorbic acid (0.1—0.8 mM) were added for a standard curve set-up. Adding of methanolic plant extracts in reaction mixture resulted in absorbance decreasing as a consequence of ABTS radical depletion. Additional sample dilution was needed for fitting the linear range of the standard curve. Absorbance alterations were read from standard curve and the TAA was expressed as micromoles of ascorbic acid equivalents (standard) per gram of fresh and dry weights.
The total phenolic content of the samples was determined according to the Folin—Ciocalteu's spectrophotometry (2501 PC, "Shimadzu", Japan) procedure using gallic acid as a standard for the calibration curve . Powdered samples were mixed with 0.25 N Folin— Ciocalteu reagents, then after 3 min 0.2 M sodium carbonate solution was added and the mixture was incubated for 60 min at 25°C temperature. Gallic acid was used for constructing the standard curve (0.1—2.0 mM). Additional sample dilution was needed for fitting the linear range of the standard curve. Results were read at 724 nm and expressed as micromoles of gallic acid equivalent per gram of fresh and dry weights.
HPLC—MS analysis of methanol extracts was performed using Dionex Ultimate 3000 UHPLC system ("Thermo Fisher Scientific", Germany) equipped with a diode array detector (DAD) and connected to TSQ Quantum Access Max triple-quadrupole mass spectrometer ("Thermo Fisher Scientific", Switzerland). Separation was performed at 30°C on Hypersil gold C18 column (50 x 2.1 mm) with 1.9 |m particle size ("Thermo Fisher Scientific", USA). The mobile phase consisted of (A) 0.1% formic acid in water, and (B) ac-etonitrile (MS grade, "Fisher Scientific UK", United Kingdom), which were applied in the following gradient elution: during first 3 min 5—20% B, 3—5 min 20— 40% B, 5.0-7.5 min from 40 to 50% B, 7.5-8.5 min 50-60% B, 8.5-10.5 min 60-95% B, 10.5-11.5 min 95%, 11.5-12.0 min from 95 to 5% B, and kept for final 3 min in 5% B. The flow rate was set to 0.4 mL/min and the column was thermostated at 30°C.
For MS analysis, TSQ Quantum Access Max triple-quadrupole mass spectrometer with heated electrospray ionization (HESI) source was used. Vporizer temperature was 450°C, with ion source settings as follows: spray voltage 4000 V, sheet gas (N2) pressure 50 arbitrary units (arb. units), ion sweep gas pressure 0 arb. units and auxiliary gas pressure 20 arb. units, capillary temperature was 320°C and skimmer offset set to 0 V. Mass spectrometry data were acquired in both positive and negative mode, in m/z range from 100 to 1000. Multiple mass spectrometric scanning modes, including full scanning (FS), product ion scanning (PIS), and neutral loss scanning (NLS) were conducted for the qualitative analysis. Collision-induced fragmentation experiments were performed using argon as the collision gas, and collision energy was set to 30 eV.
Phenolic compounds were extracted in the medium containing methanol : water : hydrochloric acid as 70 : 30 : 5. The homogenates were centrifuged at 10000 g for 20 min and supernatants were filtered through 0.22 ^m syringe filter prior to HPLC analysis.
Data analysis was performed by one way ANOVA at p < 0.05 in Excel 2003. Correlation analysis was performed in IBM SPSS v. 20.
RESULTS AND DISCUSSION
In previous paper  we suggested the role of specific catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), and polyphenol oxidase (PPO) isoforms in separate morphogenic stages during T. bellus direct shoot organogenesis (initiation of shoot organogenesis — the 10th day, meristemoids formation — the 20th day, shoot buds formation — the 40th day, and rosette fo
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