Effects of 5-aminolevulinic acid on osmotic adjustment and antioxidant system in mung bean under chilling stress

Temperature is an important factor for growth, development, productivity and geographical distribution of many plants (Nahar et al. 2015). Chilling stress is a major abiotic stress of crop production in Northeast China. Chilling stress exposure has been shown to enhance production of reactive oxygen species (ROS) and oxidative stress occurs (Nahar et al. 2015). The ROS, which include superoxide radical (O2*-), hydrogen peroxide (H2O2), hydroxyl radical (.OH), and singlet oxygen (1O2), cause damage to structural proteins, nucleic acids, enzymes, cell membranes, and other essential molecules involved in plant metabolism (Sharma et al. 2012, Nahar et al. 2015). Plants have developed mechanisms to tolerate environmental stress conditions through various physiological adaptations, including non-enzymatic and enzymatic ROS scavenging pathways (Hossain et al. 2010, Sharma et al. 2012, Nahar et al. 2015). Non-enzymic components of the antioxidative defense system include reduced ascorbate (AsA) and reduced glutathione (GSH) as well as osmotic adjustment substances as proline, soluble sugars, and soluble proteins which protect membrane integrity and cellular components from dehydration (Ozlem and Ekmekci 2011). The enzymatic antioxidants comprise superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR), etc. (Gill and Tuteja 2010, Hossain et al. 2010). These enzymes, through step-by-step reaction, scavenge ROS with AsA and GSH as electron acceptors (Gill and Tuteja 2010). Nahar et al. (2015) has established that low temperature stress increased H2O2 and MDA content. Exogenous spermidine (Spd) in low temperature treatment increases the content of AsA and GSH, decreases the content of oxidized ascorbate (DHA) and oxidized glutathione GSSG, and improves activity of APX and GR. Various strategies are being employed in order to minimize the adverse effects of environmental stresses in plants. Exogenously applied plant growth regulators (PGRs) is an effective, facile, and practical technique to enhance tolerance of crops, and this approach has been used widely in recent years. One of the PGRs is 5-aminolevulinic acid, or 5-amino-4-oxo-pentanoic acid (ALA), which has a relative molecular mass of 131, and it is an essential precursor of tetrapyrrole compounds including chlorophyll, heme, and phytochrome (Balestrasse et al. 2010) and its formation may be the rate limiting step. Hotta et al. (1997a,b) observed that low dosage of ALA has plant growth regulator properties, such as promoting chlorophyll biosynthesis and enhancing photosynthesis (Memon et al. 2009), responding to environmental stresses (Korkmaz and Korkmaz 2009, Korkmaz et al. 2010, Zhang et al. 2012, Dan et al. 2013, Fu et al. 2016), and promoting recovery of growth after herbicide applications (Zhang et al. 2008). High dosages of ALA can cause the accumulation of several chlorophyll synthesis intermediates, but also production of ROS leading to oxidative stress (Balestrasse et al. 2010). Materials and methods Plants and chilling stress: Mung bean [Vigna radiata (L.) Wilczek] cv. Lvfeng 2 was used for chilling stress experiments at National Coarse Cereals Engineering Research Center. Ten seeds were sown in plastic pots (9.0 cm lower inside diameter, 13.0 cm upper inner diameter, and 11.0 cm height) filled with mixed air-dried soil + Vermiculite + Perlite (2:1:1, v/v/v) and placed in a growth chamber under day/night temperatures of 25/18 ℃, a 12-h photoperiod, an irradiance of 300 μmol m-2 s-1, and a 75 % relative humidity. The plants were sprayed with 0.5 mM ALA solution (Balestrasse et al. 2010) and distilled water for control at cotyledon stage. About 48 h after ALA treatment, the plants were exposed to chilling stress conditions for 0, 6, 12, 24, 48, and 60 h, respectively. For temperature treatments, the indoor controlled growth chambers was already set at low temperature treatment of 5 °C and 25 °C as control. The leaves were harvested directly into liquid nitrogen and stored at -40 °C for further use. This experiment was set up in a completely randomized design with four replications. Reactive oxygen species: The production rate of O2*- was appraised according to Yang et al. (2010). Leaf tissues (100 mg) were ground in a K-P buffer solution (pH 7.8), and centrifuged at 5 000 g. To monitor the nitrite formation from hydroxylamine, the supernatant was mixed with 10 mM hydroxylamine hydrochloride and extraction buffer, and incubated at 25 °C for 20 min. After 20 min, 7 mM naphthylamine and 17 mM sulfanilamide were added and the mixture was incubated again at 25 °C for 20 min. Absorbance was measured at 530 nm (UV-3600 Plus, Shimadzu, Kyoto, Japan). Lipid peroxidation: Lipid peroxidation in leaves was measured by estimating the content of thiobarbituric acid (TBA) reactive substances, which determined malondialdehyde (MDA) content (Kaur et al. 2015). A leaf sample (0.1 g) was ground in 5 cm3 of phosphate buffer (pH 7.8) and centrifuged at 12 000 g for 20 min. To a 1 cm3 aliquot of the supernatant, 1 cm3 of phosphate buffer saline (PBS) (pH 7.8), and 2 cm3 of 20 % (m/v) trichloroacetic acid containing 0.5 % (m/v) TBA were added and incubated in boiling water for 15 min. Then the mixture was quickly cooled in an ice bath and was centrifuged at 1 800 g for 10 min. The absorbance of the supernatant was read at 532 nm and 600 nm (UV-3600 Plus). The MDA content was calculated from the extinction of absorbance of 155 mM-1 cm-1. Osmolytes determination: Total soluble sugar content of each sample was determined following the method of Buysse and Merckx (1993). Fresh leaf samples (0.1 g) were homogenized in 10 cm3 of 80 % (v/v) ethanol and extracted in a water bath at 80 °C for 15 min. The supernatant was centrifuged three times (10 000 g, 20 min). The combined liquid supernatants were concentrated in 25 cm3 tubes as a mother liquor. Total soluble sugar content was measured at 630 nm by the colorimetry of sulfuric acid-anthrone method (UV-3600 Plus). Non-enzymatic antioxidants: The estimation of AsA and oxidized ascorbate (DHA) was done according to the method of Zhang and Kirkham (1996). Plant tissue was homogenized in 5 % (m/v) phosphoric acid, and the homogenate was centrifuged at 22 000 g and 4 °C for 15 min. The mixture contained 0.5 cm3 of the enzyme extract, 1.0 cm3 of absolute ethanol, 0.5 cm3 of 0.4 % H3PO4-ethanol, 1.5 cm3 of 5 % phosphoric acid, 0.5 cm3 of 0.03 % (m/v) FeCl3-ethanol and 1.0 cm3 of 0.5 % (m/v) phenanthroline-ethanol. The mixture was boiled for 90 min in a constant temperature water bath (30 °C) and then cooled to room temperature. The absorbance was read at 530 nm (UV-3600 Plus). Antioxidant enzymes: Mung bean leaves (0.5 g) were homogenized in 10 cm3 of chilled/ice cold 50 mM PBS extraction buffer (pH 7.0) using a mortar and a pestle, and centrifuged at 11 500 g and 4 °C for 15 min. The supernatant was used for enzyme activity assays.