ФИЗИОЛОГИЯ РАСТЕНИЙ, 2015, том 62, № 4, с. 514-520
BIOCHEMICAL AND PHYSIOLOGICAL RESPONSES OF Brassica napus PLANTS TO HUMIC ACID UNDER WATER STRESS1 © 2015 R. Lotfi*, P. Gharavi-Kouchebagh**, H. Khoshvaghti*
*Department of Agriculture, Payame Noor University (PNU), Iran **Department of Plant Eco-physiology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran
Received October 22, 2014
This study examines the effects of humic acid (HA, 3 and 6 mg/L) on some biochemical and physiological parameters of rapeseed (Brassica napus L.) plants under different water supply conditions (60, 100, and 140 mm evaporation from class A pan). Water stress decreased chlorophyll a (Chl a) and total chlorophyll (ChlT) content in plants but proline content partly increased with increasing water stress severity. Plants treated by HA had more Chl a and ChlT content under both well and limited water conditions. Application of HA improved the PSII and peroxidase activity of rapeseed plants under all irrigation treatments. Ascorbate peroxidase activity under severe water stress condition was increased by 70 and 95%, compared with that under moderate and well watering conditions, respectively. Catalase activity was 51 and 69% less under well watering than that of moderate and severe water stress conditions, respectively. The highest activity of ascorbate peroxidase was recorded in plants treated by 6 mg/L HA. HA-treated plants had 42, 8.5, and 15% more soluble protein content under well watering, moderate and severe water stress conditions, respectively, compared with control plants (non-humic acid). Malondialdehyde was increased with increasing the severity of water stress, application of HA significantly reduced the amount of this trait under water stress conditions. It was shown that application of HA increased the activity of antioxidant enzymes, improved PSII activity and consequently decreased lipid peroxidation in rapeseed plants.
Keywords: Brassica napus — antioxidant enzymes — chlorophyll content — humic acid — lipid peroxidation — proline — PSII activity — water stress
Drought is considered as one of the most important environmental stresses limiting plant growth and crop productivity. Water stress can be defined as the absence of adequate soil moisture necessary for a plant to grow normally and complete its life cycle. Common plant symptoms to water deficit are stunt growth, limit CO2 diffusion to chloroplasts because of stomata closure, reduce photosynthesis rate and accelerate leaf senescence .
One of the biochemical changes occurring in plants when subjected to water stress is the accumulation of ROS (reactive oxygen species) . Chloroplasts, mitochondria, and peroxisomes in plant cells are important intracellular generators of ROS, which can be responsi-
1 This text was submitted by the authors in English.
Abbreviations'. APX — ascorbate peroxidase; CAT — catalase; Chl a — chlorophyll a; ChlT — total chlorophyll; HA — humic acid; MDA — malondialdehyde; POD — peroxidase; MWS — moderate water stress; SWS — severe water stress; WWC — well watering conditions.
Corresponding author. Ramin Lotfi. Department of Agriculture, Payame Noor University (PNU), Iran; e-mail. email@example.com
ble for the occurrence of oxidative damages under abiotic stress . Increasing evidences suggest that water stress induces oxidative stress in various plants , including both free radical (superoxide radicals, hydroxyl radical, perhydroxy radical and alkoxy radicals) and non-radical (molecular) forms (hydrogen peroxide and singlet oxygen), which can destroy proteins, lipids, carbohydrates and nucleic acids . Plants can respond to osmotic stress at morphological, anatomical and cellular levels with modifications that allow the plant to avoid the stress or to increase its tolerance . The induction of ROS-scavenging enzymes, such as superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), catalase (CAT) and other compounds such as carotenoids, ascorbic acid, thiols, soluble protein and glutathione are the most common mechanism for detoxifying ROS synthesized during stress .
Plants under water stress also produce large amounts of amino acids, such as proline for improving drought resistance. Proline prevents oxidation inside the cells under drought stress . Damage to fatty acids of membrane could produce small hydrocarbon fragments including malondialdehyde (MDA).
MDA is the final product of plant cell membrane lipid peroxidation and is important sign of membrane system injury .
The application of organic products such as humic acid is one of the methods that may reduce irrigation intervals, improve the water use efficiency and decrease the effect of drought stress on plants . Humic acid is a suspension, based on potassium humates, which can be applied as a plant growth stimulant or soil conditioner. It improves soil physical property, ion exchange capacity and water holding capacity. Therefore, it improves plant growth and helps plants to resist droughts . Furthermore, the growth promoting activity of humic substances was found to be caused by plant hormone-like material contained in the humic substances. Under water stress, foliar fertilization with humic molecules increase leaf water retention, the photosynthetic rate and antioxidant metabolism .
Rapeseed (Brassica napus) is an important agricultural crop grown primarily for its edible oil. The meal that remains after oil extraction has value as a source of protein for the livestock feed industry. In Iran, the production of the rapeseed plant is limited by soil salinity and drought. A new scheme for amelioration water stress is to overcome the irregularities in plant physiological and biochemical mechanisms by some of the natural organic materials like humic acid. Therefore, the objective of this research is to investigate the effects of foliar application of humic acid on physiological and biochemical properties of rapeseed plants under different water supply conditions.
MATERIALS AND METHODS
Plant materials and treatments. A split plot experiment (using RCB design) with three replications was conducted in 2013 at the University of Payame Noor (PNU), in order to determine the effects of humic acid (HA) application on some biochemical and physiological traits of rapeseed (Brassica napus L.) under water stress. Three irrigation treatments, including well watering conditions, moderate water stress, and severe water stress: 60, 100 and 140 mm evaporation from class A pan, respectively, were located in main plots and three levels of humic acid including non-humic acid (NHA as control), 3 (HA1) and 6 (HA2) mg/L ofhumic acid were allocated to sub plots. Seeds of rapeseed were treated by 2 g/kg Benomyl and then were sown on May 22, 2013 in 3 cm depth of a sandy loam soil. Seeding density was 90 seeds/m2. Each plot consisted of 4 rows of 4 m length, spaced 25 cm apart. All plots were irrigated immediately after sowing and after seedling establishment, plants were thinned to 65 plants/m2. Subsequent irrigations were carried out on the bases treatments. Two levels of humic acid were sprayed on plants in vegetative and early flowering stages.
Chlorophyll content. Chlorophyll content was determined using the methods proposed by Harbone .
The four fully expanded leaves in flowering stage were detached from the plants. Prior to extraction, fresh leaf samples were cleaned with deionized water to remove any surface contamination. 1 g of the leaf tissues was homogenized in 80% acetone at 4°C.
Maximum efficiency of PSII. The chlorophyll fluorescence induction parameters were measured in leaves by a chlorophyll fluorometer (0S-30, "Optisciences", USA) at flowering stages. Fluorescence emission was monitored from the upper surface of the leaves. Dark-adapted leaves (30 min) were initially exposed to the weak modulate measuring beam, followed by exposure to saturated white light to estimate the initial (F0) and maximum (Fm) fluorescence values, respectively. Variable fluorescence (Fv) was calculated by subtracting F0 from Fm. The quantum yield (Fv/Fm) measures the efficiency of excitation energy capture by open PSII reaction centers, representing the maximum capacity of light-dependent charge separation in PSII .
Enzyme assays. In flowering stage, young leaves of rapeseed plants were collected and enzyme activities were assayed. Leaf samples were collected in an ice bucket. Leaves were then washed with distilled water and surface moisture was wiped out. Leaf samples (0.5 g) were homogenized in ice-cold 0.1 M phosphate buffer (pH 7.5) containing 0.5 mM EDTA with a pre-chilled mortar and pestle. Each homogenate was transferred to centrifuge tubes and centrifuged at 4°C in Beckman refrigerated centrifuge for 15 min at 15000 g. The supernatant was used for enzyme assay.
Catalase (CAT, EC 220.127.116.11) activity. The reaction mixture (1.5 mL) consisted of100 mM phosphate buffer (pH 7.0), 0.1 mM EDTA, 20 mM H202, and 20 |L of the enzyme extract. The reaction was started by the extract addition. A decrease in the H202 content was monitored at 240 nm, quantified using its molar extinction coefficient (s = 36/(M cm)), and the results expressed as U/(mg protein min) .
Peroxidase (POD, EC 18.104.22.168) activity was estimated according to Hemeda and Klein . The reaction mixture contained 25 mM phosphate buffer (pH 7.0), 0.05% guaiacol, 10 mM H202, and the enzyme extract. Activity was determined by the increase in absorbance at 470 nm due to guaiacol oxidation (s = 26.6/(mM cm)).
Ascorbate peroxidase (APX, EC 22.214.171.124) activity was determined according to Nakano and Asada . The reaction mixture contained 50 mM potassium phosphate (pH 7.0), 0.2 mM EDTA, 0.5 mM ascorbic acid, 2% H202, and 0.1 mL of the enzyme extract in a final volume of 3 mL. A decrease i
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