ФИЗИОЛОГИЯ РАСТЕНИЙ, 2015, том 62, № 5, с. 660-667
COMPARATIVE RESPONSE OF ANNUAL Medicago spp. TO SALINITY1 © 2015 M. Karami*, F. Rafiei***, B. Shiran*, M. Khodambashi*
*Department of Plant Breeding and Biotechnology, College of Agriculture, Shahrekord University, Shahrekord, Iran **Biotechnology Research Department, Shahrekord University, Shahrekord, Iran
Received December 2, 2014
The present study was conducted to evaluate the morphological and physiological traits and the expression profile of antioxidant genes in four annual Medicago genotypes (M. truncatula Karaj; M. truncatula Qom; M. polymorpha; M. laciniata) under salinity. The experiment was carried out in a randomized complete block design, in which salinity (0 and 100 mM NaCl) allotted to main plots and genotypes assigned to subplots. The results indicated that salinity significantly increased Na+ content and decreased K+ content thereby increased the ratio of Na+/K+ in all annual Medicago genotypes. Salinity caused reduction in dry weight of shoot and root in all plants. Total chlorophyll and carotenoid contents declined by virtue of salt stress and such reduction was more remarkable in M. truncatula compared to M. polymorpha and M. laciniata. In a reverse manner, salinity increased anthocyanin content in all Medicago genotypes. Salinity augmented the overexpression of glutathione S-transferase (GST), glyoxalase I (GLXI), glutaredoxin (GRX), and peroxidase (PERX) genes in annual Medicago genotypes. Nevertheless, the overexpression of GLXI, GRX, and PERXgenes was greater in M. truncatula plants than in M. polymorpha and M. laciniata plants. The expression of antioxidant's genes in M. polymorpha and M. laciniata plants was greater than in M. truncatula and M. truncatula plants. In conclusion, M. polymorpha and M. laciniata were regarded as salt-tolerant species by less reduction in chlorophyll and carotenoid contents, less increase in anthocyanin content, and higher expression of GLXI, GRX, and PERX.
Keywords: Medicago spp. — antioxidants — glutaredoxin — glutathione — glyoxalase — peroxidase
Salinity is one of the major constraints in agriculture  which increases vulnerability of plants by means of several mechanisms including water scarcity, ion toxicity, nutrient imbalance, and oxidative stress . Oxidative stress per se harms plant constitutes including protein, lipids, carbohydrates, and nucleic acids. Plant cells are equipped with a complex of enzymatic and non-enzymatic antioxidants that work in concert to efficiently suppress oxidative stress [3, 4]. Non-enzymatic antioxidants of plants are a wide range of compounds including glutathione (GSH), carotenoids, flavonoids, and tocopherols [5, 6]. Plant enzymes associated with antioxidative and detoxifying mechanisms comprise glutathione S-transferases (GSTs), glu-
1 This text was submitted by the authors in English.
Abbreviations'. Ant — anthocyanin; Car — carotenoids; Chl — chlorophylls; Fla - flavonoids; DW — dry weight; FW — fresh weight; GRX — glutaredoxin; GSH — glutathione; GST — glutathione S-transferase; GLXI — glyoxalase I; MG — methylglyoxal; PERX — peroxidase; SOD — superoxide dismutase. Corresponding author. Fariba Rafiei. Department of Plant Breeding and Biotechnology, College of Agriculture, Shahrekord University, Shahrekord, Iran; fax. +983814424428; e-mails. Fari-ba.Rafiei@gmail.com; Fariba.Rafiei@agr.sku.ac.ir
tathione reductase, peroxidase (PERX), glutaredoxin (GRX), glyoxalase I (GLXI), superoxide dismutase (SOD), and catalase [6, 7].
There are several strategies to increase salt tolerance in economically important plants including the selection of plants under high salt conditions with subsequent breeding techniques and marker-assisted selection . Other possible alternatives are to manipulate regulation of stress-related genes especially those controlling antioxidant enzymes or to introduce novel genes by genetic engineering. Annual Medicago spp. are glycophytes that are well adapted to different environmental conditions. Among Medicago family, M. truncatula is frequently used to evaluate the response to salinity stress . The present study investigates the response of different species of annual Medicago spp. to salt stress with attempts to identify saltsensitive and salt-tolerant species. Criteria measured were indices of tolerance to salinity (dry weight, chlorophyll content, the contents of Na+ and K+, and the ratio of Na+/K+), and changes in non-enzymatic antioxidant components (carotenoids, anthocyanins, and flavonoids) along with the expression of antioxi-dant genes GLXI, GST, GRX, and PERX.
MATERIALS AND METHODS
Plant material and growth conditions. This study was conducted in a two-month trial. Seeds of Medicago laciniata, and M. polymorpha were obtained from Iranian Forest, Range and Watershed Organization (www.frw.org.ir). Seeds of M. truncatula Karaj and M. truncatula Qom were provided by Iranian Seed and Plant Improvement Institute (www.spii.ir). Karaj and Qom are the names of habitat of the plants. All seeds were scarified in concentrated sulfuric acid for 5 min and rinsed 4—5 times with distilled water. To ensure uniform germination, scarified seeds were placed in Petri dishes and stored for 1 day at 24 °C in greenhouse. Germinated seedlings were transferred to germination trays containing a mixture of coco peat : per-lite in 70 : 30 ratio, and grown in greenhouse. After a week, the plantlets were transferred to specific containers (40 x 50 x 25 cm) filled with Hoagland solution. Hoagland medium used to irrigate the plantlets was gradually concentrated on weekly basis so that its concentration was 25% in the week 1 and reached 100% in the week 3.
Subsequently, salt stress treatment (100 mM NaCl) was applied in a four-week-period trail. Salt was dissolved in Hoagland solution to provide a saline solution with the concentration of 100 mM NaCl. The control plant samples were not exposed to salinity. Each container had triplicates of all four Medicago genotypes. None of the genotype was nodulated. One month after salinity exposure, seven plants were sampled from each replicate (21 plants per treatment) to measure weights of dried shoot and dried root as well as physiological traits including contents of chlorophyll (Chl), carotenoids (Car), anthocyanins (Ant), and Na+ and K+ in shoots and roots.
Dried tissues (DW) were used for measuring contents of Na+ and K+ in the digested samples using flame photometer.
Photosynthetic pigments (Chl) were quantified spectrophotometrically in acetone extracts and calculated as mg/g fresh weight (FW) . The content of the carotenoids (Car) was calculated as mg/g FW using the Lichtenthaler equations .
Anthocyanins (Ant) were extracted from oven-dried tissue samples of leaves. In brief, 0.1 g of dried powder was suspended in 10 mL volume of acidified methanol (methanol : H2O : HCl, 79 : 20 : 1, v/v) and auto-extracted at 0°C for 72 h. After centrifugation, the absorbance was measured at 530 and 657 nm for each supernatant. Anthocyanin content was calculated by formula :
Ant = (4530 - 1/3 A657)/g DW.
Flavonoid (Fla) also was expressed as absorbance at 300 nm and Fla content calculated as A300/g DW .
RNA extraction, cDNA synthesis and RT-PCR. Samples of roots and leaves for transcriptome analysis were harvested 24 h after salinity treatment. Each sam-
ple had two biological replicates. Total RNA was extracted by RNA isolation kit ("DENAzist Asia Co.", Iran) according to the manufacturer's instructions. The purified total RNA was quantified spectrophoto-metrically (WinASPECT® PLUS, "Analytik Jena", Germany). The quality of RNA was checked on RNA MOPS gel. DNase treatment was carried out by Fermentas Kit ("Fermentas", USA). The first strand complementary DNA (cDNA) was synthesized using the M-MuLV reverse transcriptase kit ("Vivantis") with 1.5 |g of total RNA as a template, and 1|L oligo dT18 (40 |M). Prior to use in qRT-PCR reaction, cDNAs were diluted to 1/2. qPCR reactions (10 |L) included 2 |L cDNA synthesis product, 200 nM primer and 5 |L SYBER Premix Ex Taq II ("TaKaRa") using a ROTOR-Gene®Q Real-Time PCR System ("Qiagen"). Each biological replicate had four technical replications in RT-PCR.
In the current study, the antioxidant genes including GST, GLXI, GRX, and PERX were selected based on analysis of microarray data, GSE14029 including GSE13921 and GSE13907 (available in NCBI). These microarray data conducted by Li et al.  and included data from transcriptome analysis of M. truncatula cv. A17 in salt stress condition. The relevant TAIR annotation (homolog genes in Arabidopsis) for these genes, obtained using Plant Expression Database (www.plexdb.org/mod-ules/gl Suite/gl_main.php). FASTA sequence of the mRNA encoding GST, GLXI, GRX, and PERX were downloaded from PLEXdb and blasted against M. truncatula genome in NCBI (BLASTN).
Using NCBI database, mRNA sequence of XM_003623148.1 (923 bp) was selected as candidate gene for GST, and BT146528 (953 bp) having GLXI as conserved domain, was selected as candidate for GLXI. XM_003602413.1 (1023 bp) and XM_003636842.1 (1298 bp) considered as candidates for PERX, and GRX, respectively. Specific primers were designed for each gene by means of Beacon Designer Software (table 1). Before RT-PCR step, normal PCR was used for checking the specificity of the designed primers. The efficiency of designed primers, and melting curve were also determined. Mt Actin considered as internal control gene in RT-PCR.
Statistical analysis. Morphological and physiological data were subjected to randomized complete block design (RCBD) using SAS software (v. 9). The statistical model for morpho-physiological data was as follows:
Yijk = ^ + Ri + Aj + ARij + Bk + ABjk + eijk.
In this model, Yj is observation; ^ - the general mean; Ri - the effect of block; Aj - the effect fo
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