1

Abscisic and jasmonic acid, increase antioxidant enzymes in Thevetia peruviana callus

2Jack Rincn-Prez1, Federico A. Gutirrez-Miceli1, Daniel Gonzlez-Mendoza2

31Instituto Nacional de Tuxtla Gutirrez, Departamento de Biotecnologa vegetal, Tuxtla Gutirrez
429000, Chiapas, Mxico
52Instituto de Ciencias Agrcolas, Universidad Autnoma de Baja California, Ejido Nuevo Len
621705, Baja California, Mxico
7*Corresponding author:
8
91

Introduction

10Plants naturally occurring reactive species scavenging that are cytotoxic and harmful to nucleic
11acids, proteins and cell membrane through antioxidant enzymes activation that are involved in
12cell detoxification (Chakraborty et al., 2015). However be are beginning to use technologies that
13induce the activation of this enzymes type to make tolerant plants to different stress factors.
14Elicitation is an alternative since that elicitor effects in plants affect the production of phenolics
15and activation of several enzymes related to defense mechanisms (Thakur and Sohal, 2013).
16Recent researches (Guo et al., 2012) demonstrate that abscisic acid (ABA) increases antioxidant
17enzymes activity doing plants more tolerant to different stress factors. Likewise be demonstrate
18that exogenous jasmonic acid (JA) increases antioxidant enzymes activity (Parra-Lobato et al.,
192009) the which are relate to different biotic and abiotic stress. Knowing this is important apply
20in plants to which can get some benefit as it is T. peruviana, which is a inedible plant, that occurs
21wild in Mexico, and researchs recent have revolved around the clinical, pharmacologic aspects,
22and recent decades it has been studied the possibility that T. peruviana can be an oilseed crop for
23biodiesel production (Sahoo et al., 2009).
24Studies of antioxidant enzyme activation in elicited callus is a topic little discussed, is why the
25aim of this work was to evaluate the effect of JA and ABA on antioxidant enzymes activity in T.
26peruviana callus.
27
28Keywords: Peroxidase, catalase, proline, phenylalanine ammonia-lyase.
29
30
31

322

Materials and methods

33
341.1

Plant materials and establishment of callus

35Randomly fruits of different plants of T. peruviana were collected from road divider Highway,
36Chicoasen, Tuxtla Gutirrez, Chis, Mxico (16.760728, -93.146907) during the months of
37October and November 2014. The fruits collected were washed and cut with secateurs so that the
38seed remained uncovered and embryos with cotyledons were selected as explants. In tissue
39culture hood was performed disinfection with commercial sodium hypochlorite 5-6% (v/v) for 10
40min and 70% ethanol (v/v) for 10 min, with three rinses with sterile distilled water after each
41disinfectant. Calli were obtained from embryos with cotyledons on SH medium supplemented
42with plant growth regulators (PGR) 2 mg/L 2,4-D y 0.5 mg/L KIN (Zabala et al., 2010), 3%
43sucrose and 0.3% phytagel. The pH of the medium was adjusted to 5.8 0.1 prior to autoclaving
44at 15 psi and 121 C for 15 min Cultures were kept in the growth chamber at 22 C under
45constant light conditions (white fluorescent light, intensity 45.8 M m-2 s-1), and regularly
46subcultured every 4 weeks on the same medium and under the same conditions for three-months
47until get old friable callus.
48
491.2

Elicitor treatments

50Stock solutions of JA and ABA were prepared following Sigma-Aldrich provider instructions by
51dissolving 96 % (v/v) methanol. Friable callus were transferred to glass jars containing 20 mL
52fresh medium without PGR but supplemented with JA and ABA following the experimental
53design (Table 1), samples were taken to 7 and 9 days after incubation with the elicitors.
54
55Table 1 Design to evaluation JA and ABA effect on antioxidant enzymes activity and proline

Tiempo (das)
7

9
56

Tratamiento
Control 7
1
2
3
Control 9
1A
2A
3A

JA (M)
0
75
0
75
0
75
0
75

ABA (M)
0
0
55
55
0
0
55
55

572.1

Proline content

58Proline content was determinate in accordance to Slabbert and Krger, (2014) with modifications.
59Briefly, 0.04 g liophylized callus was homogenized with 3% (w/v) sulfosalicylic acid and
60centrifuged at 4000 rpm for 10 min. A 200 l aliquot of the supernatant was mixed with 400 l of
61the reagent mixture (glacial acetic acid, phosphoric acid and ninhydrin) and heated in sealed test
62tubes at 100 C for 1 h. After cooling down, 2 ml toluene was added to each sample. Proline
63content was read spectrophotometrically at 520 nm and expressed as moles per gram dry weight
64(DW).
65
662.2
672.2.1

Antioxidant enzyme activity

Peroxidase (POD)

68The POD assay was carried according to Tejacal et al., (2005), method using guaiacol as a
69substrate, in a reaction mixture (3 mL) containing 2.6 mL of 100 mM sodium phosphate buffer
70(pH 7.1), 0.25 mL of 0.1 M guaiacol, 0.1 mL of 0.25 % H 2O2 and 0.05 mL of supernatant. The
71increase in absorbance at 470 nm due to the guaiacol oxidation was recorded for 3 min. One unit
72of enzymatic activity was defined as the amount of the enzyme that caused a change of 0.01 in
73absorbance per minute. The POD activity was expressed as U. min-1 g -1 DW.
74
752.2.2

Catalase (CAT)

76Catalase activity was determined by consumption of H 2O2 using the method of Tejacal et al.,
77(2005). The reaction mixture (3 mL) contained 10 mM Tris-HCl buffer pH 8.5, 0.88 % 0.1 mL
78H2O2 in 100 mM Tris-HCl and 0.1 mL enzyme extract. The reaction was initiated by adding the
79H2O2. The consumption of H2O2 was monitored spectrophotometrically at 240 nm for 2 min.
80Enzyme activity was expressed in M H2O2 min1.
81
822.3

Phenylalanine ammonia lyase (PAL)

83PAL activity was determined according Ivanov et al., (2013) with modifications. Briefly samples
84of 0.4 g were homogenized in 15 mL borax buffer (100 mM, pH 8.8) and mercaptoethanol (20
85mM) for 1 h at 1 C. Homogenates were centrifuged (12000 rpm for 15 min to 4 C) and
86supernatants were collected. Was extracted 6.5 mL of the volumes of supernatants and was added
87ammonium sulfate 1.76 g and agitated for 30 min at 40 C. Subsequently was centrifuged at

8812000 rpm for 20 min. The precipitated was resuspend in a solution of ammonium acetate and
89ammonium acetate (4.5 mL) and then was leave in incubation to 40 C for 2 h. 2 mL of the
90investigated extracts were added to 3.4 mL peroxide and 0.6 L 0.1 M l-phenylalanine. Formed
91cinnamic acid was measured after 0 and 120 min spectrophotometrically at 290 nm, PAL activity
92was expressed in m cinamic acid/min (m CA/min).
93
942.4

Statistical analysis Significance

95Significance of treatment effects was determined by using variance analysis (ANOVA).

96Variations between treatments means were analyzed using Tukeys test (p 0.05).
97
983

Results

99
100Table 2 Effect of JA and ABA on proline content and PAL activity

PAL (m CA/min)
Treatment
Proline (g/g)
0,0260,0004 a
0,0020,000 b
Control 7
0,0190,0002 c
-0,0240,000 e
T1
0,0180,0003 d
-0,0420,004 g
T2
0,0190,0003 c
-0,0090,003 c
T3
0,0170,0003 e
0,0000,000 b
Control 9
0,0250,0000 b
-0,0340,000 f
T1A
0,0190,0002 c
0,0540,003 a
T2A
0,0150,0002 f
-0,0140,000 d
T3A
101Values designated by the different letters are significantly different (p 0.05).
102
103
104
105
106
107Table 3 Effect of JA and ABA on antioxidant enzymes

Treatment
Control 7
T1
T2
T3
Control 9

POD (U/g dw)

11,71
58,90
66,25
11,11
148,20

CAT

T1A
T2A
T3A
108

219,23
86,52
15,37