ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 1, с. 59-68
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
УДК 581.1
ANTIOXIDANT ENZYME RESPONSES TO SALINITY STRESS OF Jatropha curcas AND J. cinerea AT SEEDLING STAGE1
© 2014 M. Hishida*, F. Ascencio-V&He*, H. Fujiyama**, A. Orduño-Cruz*, T. Endo**,
J. Á. Larrinaga-Mayoral*
*Centro de Investigaciones Biológicas del Noroeste, Calle Instituto Politécnico Nacional, Col. Playa Palo de Santa Rita, La Paz, Mexico **Faculty of Agriculture, Tottori University, Tottori City, Japan Received October 15, 2012
The salt-sensitive humid tropical biodiesel crop, Jatropha curcas, was subjected to a 28-day exposure to salinity (0, 50, 100, and 200 mM NaCl), and activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX), the rate of lipid peroxidation, stomatal conductance, mineral contents, and chlorophyll (Chl) content were compared to corresponding characteristics of J. cinerea, a related wild species of saline-dry areas. Biomass production decreased under the influence of 50 mM NaCl in both species, and the reduction was larger in J. curcas than in J. cinerea at higher NaCl concentrations. In both species, stomatal conductance and transpiration decreased, and leaf temperature and Na+ concentration increased under salt treatment; salinity effect was stronger in J. curcas. Chl degradation enhanced only in J. curcas. In both Jatropha species, SOD, CAT, and POX activities increased with salinity. J. curcas showed higher antioxidant activity than J. cinerea. Lipid peroxidation was observed only in J. curcas at concentrations above 100 mM NaCl, partially due to a greater reduction in stomatal conductance and/or the poor ROS-scavenging system. Thus, J. cinerea had more favorable characteristics to adapt to saline environments, and young J. curcas plants could adapt to salt-affected land if soil salinity was moderate (about 50 mM NaCl in solution).
Keywords: Jatropha curcas - J. cinerea - antioxidant enzyme - salinity - stomatal conductance - reactive oxygen species
DOI: 10.7868/S0015330314010060
INTRODUCTION
Salinity is one of the major abiotic stress factors limiting plant growth and productivity, particularly in arid and semi-arid lands. It was estimated that 6% of the world's land was affected by salinization, and 30% of agricultural land was classified as degraded [1]. Salinity stress exposes plants to osmotic and ionic stresses and to secondary effects, such as oxidative stress [2, 3]. Plants show morphological, physiological, and biochemical changes, such as stomatal closure, osmotic adjustment, ion exclusion and isolation, and an increase in
1 This text was submitted by the authors in English.
Abbreviations'. CAT - catalase; Chl - chlorophyll; AT - leaf-to-air temperature gradient; E - transpiration rate; gs - stomatal conductance; NBT - nitroblue tetrazolium; PMSF - phenylmethyl-sulfonyl fluoride; POX - peroxidase; PVP - polyvinylpyrrolidone; SOD - superoxide dismutase; Ta - air temperature; Tl - leaf temperature; TBARS - thiobarbituric acid-reactive substances. Corresponding author. Juan Á. Larrinaga-Mayoral. Centro de Investigaciones Centro de Investigaciones Biológicas del Noroeste, Calle Instituto Politécnico Nacional 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S. 23096, Mexico. Fax. 52-612-125-3625; e-mail. larrinag04@cibnor.mx
the antioxidant enzyme activities to reduce the salinity effect.
The reduction of CO2 availability and inhibition of carbon fixation due to stomatal closure under salinity stress exposes chloroplasts to excessive excitation energy, which in turn could increase the generation of reactive oxygen species (ROS) causing photoinhibition and photooxidation damage therein [4]. Insufficient energy dissipation during photosynthesis could lead to the formation of a chlorophyll (Chl) triplet state that can transfer its excitation energy onto O2 to make singlet oxygen (1O2) [5] or impair electron transport and
increase the leakage of electrons to O2 forming O2-[6]. These ROS are highly reactive, and in the absence of any protective mechanism, they can seriously disrupt normal metabolism by damaging photosynthetic pigments [7], lipids, proteins, and nucleic acids through oxidative stress [8]. Antioxidant enzymes are the most important components in ROS scavenging. Superoxide dismutase (SOD; EC 1.15.1.1) is a major
scavenger of superoxide (O2 ), and its enzymatic ac-
tion results in the formation of H2O2 [9]. H2O2 is then scavenged by catalase (CAT; EC 1.11.1.6) and a variety of peroxidases (POX; EC 1.11.1.7).
Jatropha curcas (Barbados nut, physic nut) is a tropical and subtropical perennial succulent shrub in Mexico and Central America [10], where annual precipitation is between 500 and 1000 mm [11]. J. curcas received much attention as a source of renewable energy from its oily seeds (27-40%), which are easily converted into biodiesel that meets American and European standards [12]. Because of its drought-resistant characteristic [10], it is an alternative crop in dry marginal lands where it is not suitable for most plants with agricultural potential.
The arid northwestern area of Mexico has a dry subtropical climate (annual precipitation <200 mm) with frequent long dry periods. Here, J. cinerea (Arizona nettlespurge, ashy jatropha, ashy limberbush, lomboy), a related wild species, grows on saline soils along the coast and rocky areas. J. cinerea can withstand long droughts and flowers during the rainy season (June to October). Its succulence and bark have been used in traditional medicine.
Therefore, it is important to further understand the salinity effects on J. curcas. Salinity is one of the major factors limiting J. curcas production in semi-arid and arid regions, especially due to oxidative damage and suppression of antioxidant enzyme activities. In this study, the effects of salinity stress on growth, antioxidant enzyme activities, stomatal conductance (gs), and mineral contents in young J. curcas plants were measured to gain information for assessing its capacity as an alternative plant in salt-affected soils by comparing it with J. cinerea as a wild species well-adapted to drought and salinity.
MATERIALS AND METHODS
Plant material and growth conditions. Jatropha curcas seeds from Papantla, Veracruz, Mexico and J. cinerea seeds from a field near La Paz, Baja California Sur, Mexico were sterilized with 0.5% sodium hypochlorite for 10 min and rinsed three times with distilled water. The seeds were wrapped with wet paper towels at 25 °C in the dark for 14 days to stimulate germination. Seedlings were placed in 4-L pots containing 3 L of 50% Hoagland solution [13] for hydroponic cultivation. NaCl treatments (0 (control), 50, 100, and 200 mM) started 14 days after seedlings were transplanted to the pots. Four seedlings contained in each of the three replicate pots for each four treatments (n = 3) were cultivated under salt treatments in a naturally illuminated greenhouse for 28 days. Average temperature during cultivation was 22.5°C. The solution was continuously aerated, and the pH was adjusted to 5.0 using dilute H2SO4 (1 M) and NaOH (1 M); the solution was replaced every 7 days.
Growth analysis. Plants were harvested at the start of the treatment and 28 days later. Harvested plants were washed with distilled water to remove dust and other residues. Then, plants were separated into leaves, stems, and roots to measure fresh weight. After that, leaf area was measured by a portable LI-3000A leaf area meter ("LI-COR Biosciences", United States). All parts were dried at 70°C in an oven for 48 h to measure dry weight.
Measurement of stomatal conductance, transpiration rate, and chlorophyll content. Diffusive stomatal resistance, transpiration rate (E), and leaf temperature (T) of the third leaf from the shoot apex were measured with a LI-1600 porometer ("LI-COR Biosciences") at the greenhouse 14 days after treatment. Stomatal conductance (gs) was calculated as the reciprocal diffusive stomatal resistance. The leaf-to-air temperature gradient (AT) was calculated by subtracting air temperature (Ta) from Tl. Measurements were implemented twice, in the morning (between 09:00 to 10:00 UT) and at midday (12:00 to 13:00 UT). The mean temperature was 21.2 ± 0.8°C (morning) and 32.6 ± 0.4°C (midday), and mean photosynthesis active radiation (PAR) was 388 ± 72 ^mol/(m2 s) (morning) and 594 ± 46 ^mol/(m2 s) (midday).
The leaf chlorophyll (Chl) content was determined using a SPAD-502 Chl meter ("Minolta Camera", Japan) in the third leaf from the apex every 7 days.
Mineral analyses. Mineral analyses were performed with oven-dried leaves, stems, and roots. Concentrations of Na+ and K+ were determined by atomic absorption spectrophotometry (AA660, "Shimazu", Japan) after digestion with three acids (H2SO4 : HNO3 : HClO4 at a ratio of 1 : 4 : 10).
Antioxidant enzyme activities. The activities of superoxide dismutase (SOD), catalase (CAT), and per-oxidase (POX) were determined in the fully expanded third leaf from the apex at the 21 day after treatments. Leaf segments were soaked immediately in liquid N2 and stored at -80°C until analysis.
For analyzing SOD and CAT activities, leaf segments (0.2 g) frozen in liquid nitrogen were homogenized in 2 mL of ice-cold phosphate buffer (1 M KHPO4, pH 6.8), 0.2 g of polyvinylpyrrolidone (PVP), and 20 ^L of phenylmethylsulfonyl fluoride (PMSF, 1 ^M) with a mortar and pestle. The homoge-nate was centrifuged at 10 620 g at 4°C for 10 min, and the supernatant was collected to determine SOD and CAT activities.
Total SOD activity was measured following the procedure described by Suzuki [14]. The xan-thine/xanthine oxidase system was used to generate
O2 - , which reacts with nitroblue tetrazolium (NBT). The product of this reaction can be detected spectro-photometrically in 5 min (Jenway 6505, "Jenway", United Kingdom). One unit of SOD activity was de-
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