Comparative studies on tolerance of Medicago truncatula and ...

Planta (2011) 234:445–457 447soaked in concentrated sulfuric acid for approximately7 min and then thoroughly rinsed with tap water. The seedswere transferred to Petri plates at 25°C in dark for 2 daysuntil most of the seeds germinated. The uniform seedlingswere put into pots filled with vermiculite under conditionsof 23°C and 12 h of light rhythm. Plants were watered with1/2 MS solution at 3 days interval. Three-week-old seedlingswere used to study cold acclimation and freezingtolerance throughout this study. Cold acclimation wasconducted by exposing Medicago seedlings to low temperature(4°C, 12 h of light) for varying periods.Determination of survival rateThe survival rate of M. truncatula and M. falcata wasdetermined after 1 week from treatments at designedtemperatures for 5 h with cold acclimation for varyingperiods. The plants that survived the freezing treatmentscan be distinguished from the dead plants (Fig. S1).Assay of electrolyte leakageElectrolyte leakage was assayed according to the method ofPennycooke et al. (2008) with some modifications. Briefly,tubes containing three leaf discs detached from 3-week-oldplants acclimated for varying periods at 4°C were placed ina low-temperature bath. After equilibrium at 0°C for 1 h,ice chips were added to each tube. The bath temperaturewas lowered at the rate of 2°C h -1 . The tubes wereremoved at defined temperatures and thawed overnight at4°C in the dark and 6 mL of deionized water was added toeach tube. After shaking with a speed of 200 rpm at 25°Cfor 2 h, electrical conductivity was first determined (C1),and thereafter the samples were autoclaved at 121°C for20 min. After cooling to room temperature, the conductivitywas determined again (C2). Relative ion leakage wasexpressed as percentage of the total conductivity afterheating at 121°C, i.e., relative ion leakage % = C1/C2 9 100.Determination of proline (Pro) contentThe Pro content in leaves of M. truncatula and M. falcatawas determined as described by Bates et al. (1973). Briefly,leaves were harvested, weighed and extracted in 3% sulfosalicylicacid. An aliquot of each extract (2 mL) wasincubated with 2 mL of ninhydrin reagent (2.5% [w/v]ninhydrin, 60% [v/v] glacial acetic acid, and 40% 6 Mphosphoric acid) and 2 mL of glacial acetic acid at 100°Cfor 40 min, and the reaction was terminated in an ice bath.Toluene (4 mL) was added, followed by vortexing andincubation at 23°C for 24 h. The absorbance was measuredat 520 nm.Measurements of soluble carbohydratesIndividual sugar contents in the leaves of M. falcata and M.truncatula were measured by high-pressure liquid chromatography(HPLC) (Agilent 1260 Infinity). The sampleswere prepared following the protocols described by Castonguayet al. (1995). Briefly, approximately 0.5 g leavesof M. truncatula and M. falcata were weighted and groundin liquid nitrogen, and 5 mL of methanol–chloroform–water (MCW,12:5:3, by vol.) was added immediately.After vortexing, the tubes were subsequently centrifugedfor 10 min at 1,000g and the supernatant was collected.The pellet was re-suspended twice by vortexing in 5 mL ofMCW (12:5:3, v/v), centrifuged for 10 min at 1,000g andthe supernatants were combined. At the completion ofextraction, 4 mL of water and 1 mL of chloroform wereadded to the 15 mL extracts for phase separation and thetubes were centrifuged for 10 min at 1,000g. The aqueousphase was collected and evaporated to dryness on a rotaryevaporator, re-suspended in double distilled water and filteredthrough a 0.45-lm membrane before analysis byHPLC. Quantification of fructose, glucose, lactose, raffinoseand stachyose was made by comparing with theirrespective standard solution containing 50 lg lL -1 sugarspurchased from Sigma.Determination of osmolalityLeaves of Medicago seedlings were detached and put into1.5-mL tubes, and then the tubes were frozen in liquidnitrogen. The frozen leaves were thawed at room temperatureto disrupt the cell structure and then centrifuged at aspeed of 10,000g for 1 min to isolate the cell sap asdescribed by Zhang et al. (1996). The osmolality of cell sapof leaves was determined using a VAPRO 5520 vaporpressure osmometer.Determination of enzyme activitySucrose phosphate synthase (SPS, EC 2.4.1.14) activitywas assayed following the protocols of Guy et al. (1992).Briefly, extracts were prepared from leaves of M. truncatulaand M. falcata by grinding in liquid nitrogen. Theextract buffer contained 50 mM Mops-NaOH (pH 7.5),10 mM MgCl 2 , 1 mM EDTA, 5 mM DTT and 0.1% TritonX-100 (v/v). The crude extracts were centrifuged at15,000g, 4°C for 20 min, and then the supernatants werecentrifuged again before being used for determiningenzyme activity. SPS activity was measured by monitoringthe formation of sucrose-6-P synthesized from fructose-6-Pand UDP-glucose as described by Guy et al. (1992).Sucrose synthase (SuSy, EC 2.4.1.13) was assayed in thesynthetic direction by measuring sucrose formation from123