ФИЗИОЛОГИЯ РАСТЕНИЙ, 2015, том 62, № 5, с. 675-683
^_ЭКСПЕРИМЕНТАЛЬНЫЕ ^^^^^^^^^^^^
СТАТЬИ
УДК 581.1
SALINITY EFFECTS ON PIGMENTS OF Turnera diffusa (Willd.) IN VITRO1 © 2015 L. Alcaraz-Meléndez, L. A. A. Soriano-Melgar, H. G. Ayala-Castro
Agricultura en Zonas Áridas, Centro de Investigaciones Biológicas del Noroeste (CIBNOR), La Paz, Baja California Sur, México Received September 4, 2014
Salinity is an unfavourable factor to plant growth, development, and quality because high salinity increases oxidative stress and damage in plants. Damiana (Turnera diffusa Willd.) plants grow in arid zones. They are socio-economically important as they have industrial potential. Currently, no reports on their tolerance to salinity are available. This study was performed to determine the effect of salinity in damiana plants in an in vitro model under controlled conditions by applying NaCl in three levels (0.1, 1.0, and 1.5%) in Murashige-Skoog medium. To prove decrease in salinity stress, exogenous salicylic acid (SA) treatments (1 and 10 ppm) were tested, and biomass and water content were determined. Photosynthetic pigments and their degradation were determined with high performance liquid chromatography to assess the degree of salinity damage. To define the adaptation mechanism to salinity, specific peroxidase (POX) activity was quantified by spectrophotometry assay. Results showed that the determined parameters (chlorophyll a, chlorophyll b, P-carotenes, pheo-phytin, violaxanthin, and zeaxanthin contents) reduced progressively with increase in salinity level. Chlorophyllide a and pheophorbide a contents were not modified by salinity stress but responded to SA presence. POX activity increased in all treatments with plants supplemented with NaCl. Treatments by SA did not modify the negative effects of NaCl on the photosynthetic pigments, but even so significantly enhanced POX activity compared with the untreated stressed plants (NaCl without SA). The results showed that damiana plants were moderately salt-tolerant and could be grown in soils with such characteristics.
Keywords: Turnera diffusa — photosynthetic pigments — xanthophylls cycle — salicylic acid — NaCl effects — tolerance to salinity
DOI: 10.7868/S0015330315050036
INTRODUCTION
Salinity is an important environmental problem that produces negative effects and diverse alterations on plant physiology, biochemistry, development, growth, productivity, and production quality by osmotic stress and ion toxicity [1]. Salinity causes many effects on plants; negative effects include alteration and decreased growth of the different organs, physiology damage, changes in osmotic pressure, loss of photosynthetic pigments reducing plant photosynthesis, inhibition of seed germination, breach of stomatal regulation, senescence, and even death [2, 3]. Other negative effects are the increase of reactive oxygen species (ROS) formation
1 This text was submitted by the authors in English.
Abbreviations'. APX — ascorbate peroxidise; CAT — catalase; POX — peroxidases; SA — salicylic acid; SOD — superoxide dis-mutase.
Corresponding author. Lilia Alcaraz-Meléndez. Agricultura en Zonas Áridas, Centro de Investigaciones Biológicas del Noroeste, S. C. (CIBNOR), Av. Instituto Politécnico Nacional 195, Col. Playa Palo de Santa Rita Sur, La Paz, Baja California Sur. C.P. 23096. México; fax. +52 (612) 125-3625; e-mail. lalcaraz04@cibnor.mx
and oxidative stress, but plants show tolerance mechanisms to salinity stress by decreasing ROS production and increasing antioxidant enzyme activities such as superoxide dismutase (SOD), catalase (CAT), and peroxidases (POX) [3]. Plant tolerance to salinity depends on numerous factors, for example, concentration, application, and time of exposure to NaCl, plant genotypes, variety, and species, environmental and climate conditions, plant tissues, growth, and so on [4].
Plants have important signal molecules to mitigate and generate salinity tolerance; many researchers have focused on using micorrhizal fungi or growth regulators to decrease the harmful effects of salinity stress, such as salicylic acid (SA) [5]. SA is a phenolic compound produced by plants, which improves growth and development to respond to environmental stresses by reducing toxic ion accumulation [4] helping to reverse the negative effects of salinity stress and enhancing the functions of photosynthetic pigments and antioxidant enzymes in some plants [6]. Therefore, SA was employed as an exogenous treatment to help plants to induce salinity tolerance [5, 7].
Crop production in arid and semi-arid lands requires germ plasm resistant to stress, which emphasizes the need to search for alternatives to improve productivity in regions with salinity problems by identifying and characterizing new genetic resources for increase tolerance to salt stress. Damiana (Turnera diffusa Willd.), belonging to the family Turneraceae, is a shrub that grows wild in the West Indies, South America, the United States, and Mexico [8] in arid and semi-arid climates, where xerophilic shrubs and deciduous forests grow on sandy soils. Damiana is a drought tolerant plant and usually develops in soils with low NaCl concentrations and electrical conductivity of 0.84 mm-hos/cm at 20 cm in depth and 1.9 mmhos/cm at 60 cm in depth with pH from 6.00 to 6.75.
Damiana is socio-economically important because its leaves and stems are used in herbal medicine as liqueur flavouring, brewing beverage, nerve stimulator, and diuretic, and it is also considered aphrodisiac [9]. Damiana could be a good alternative for cultivation on arid lands, but its tolerance to salinity has not been assessed so far. Therefore, our work aims to evaluate salinity tolerance and the effect of NaCl at different concentrations in damiana plants in vitro by applying two different concentrations of SA to observe its role in damiana growth, peroxidase activity, as well as photo-synthetic and xanthophyll cycle pigment contents to understand the mechanisms of defence.
MATERIALS AND METHODS
Plant materials. The experiments were designed in vitro conditions because it is possible to control all variables, and thus the results would be due to salinity effects only. Damiana (Turnera diffusa Willd.) explants were the wild plants from arid zones and introduced to tissue culture. These plantlets were grown in vitro for a year to generate a homogeneous culture and used for the different treatments after they had been transferred to new medium for a month. Culture conditions were described earlier by Alcaraz-Melendez et al. [10]; medium contained 0.8% agar and 3% sucrose, without growth regulators, and the culture was cultivated in 120 mL glass jars with 20 mL of MS medium.
Treatments. Treatments were carried out with the explants in the in vitro model. NaCl and/or SA were added to MS medium to maintain the same treatment level in all plants and used as follows: (A) MS medium with NaCl (0.1, 1.0, and 1.5%) without SA; (B) MS medium with NaCl (0.1, 1.0, and 1.5%) and SA (1 ppm, "Sigma", USA); and (C) MS medium with NaCl (0.1, 1.0, and 1.5%) and SA (10 ppm) to observe SA effects with respect to NaCl concentrations. Then, plantlets were placed under light and temperature controlled conditions (25 ± 2°C, 70% relative humidity, a photoperiod of 24-hour light intensity of 100 ^E/(m-2 s)). These conditions were employed with success for damiana in vitro [10]. At
each treatment we used five flasks, one plant per flask in triplicate.
Shoot growth. Plantlet biomass was determined by fresh and dry weight at 0, 7, 15, 23, and 31 days. For fresh weight quantification, plants were weighed immediately, and dry weight was determined by drying plants in the oven at 40°C for 24 h. Shoot water content (%) was calculated by determining the difference between fresh and dry weight. Five individual plants were randomly selected per treatment in each determination.
Peroxidases and protein determination. Peroxidases (POX) were determined according to Bergmeyer's [11] method. Lyophilized leaves (50 mg) embedded 1.5 mL of phosphate buffer (0.1 M, pH 7) and homogenized with polytron (PT 3100, "Kinematica AG", Switzerland). The tubes were placed on ice, and the mixture was micro-centrifuged (refrigerated micro-centrifuge, Hawk 15/05, "Sanyo/MSE", Japan) at 1200 rpm and 5°C for 10 min. In a cell, we added 3 mL of phosphate buffer (0.1 M, pH 7), 0.05 mL of guaiacol (20.1 mM), 0.1 mL of sample, and 0.03 mL of hydrogen peroxide (12.3 mM). Readings were performed at 436 nm with Spectro Master (Model 415, "Fisher Scientific", USA) every 30 s for 120 s. One unit of POX activity was defined as the amount of POX enzyme required to increase absorbance at 0.1. Samples were taken at 3, 7, 11, 15, 19, 23, 27, and 31 days.
Proteins were determined by the method of Lowry et al. [12]. Milled and lyophilized plantlets (50 mg) and phosphate buffer (50 mM, 1.5 mL) were homogenized with polytron (PT 3100), and micro-centrifuged at 1200 rpm and 5°C for 10 min. A standard curve of bovine serum albumin (50—250 ^g/mL) was used to identify proteins.
Determination of photosynthetic and xanthophyll cycle pigments. Pigments were determined according to Vidussi et al. [13]. Liquid chromatography (HPLC, model 1100, "Hewlett Packard", Germany) was employed using the Vidu 98 program to identify pigments taking into account spectrum retention time and pattern obtained from a library of the same program. For pigment extraction, 2 mg of lyophilized and milled plantlets were taken and placed in acetone (HPLC grade 100%). Samples were centrifuged at 4000 rpm and 5°C for 15 min. Extracts were filtered through a glass fiber membrane (with a pore size of 0.45 ^m). The liquid was recovered in Eppendorf vial and stored at —20°C in the dark. Subsequently 20 ^L were taken and injected into the HPLC equipment. To separate mobile phase pigments, two solutions were used: solution I was a mixture of methanol : 1 N ammonium a
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