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The free proline contents of alfalfa plants were measured spectrophotometrically as previously described [34, 35]. Leaf tissues were homogenized in 1.5 ml of aqueous sulfosalicylic acid (3%), and the residue was removed by centrifugation at 12,000 g for 10 min. One milliliter of the supernatant with 1 ml of acid-ninhydrin and 1 ml of glacial acetic acid was boiled in a water bath at 100C for 1 h. After cooling the reaction mixture, 2 ml of toluene was added, and the mixture was vortexed vigorously and incubated at room temperature for 30 min until it separated into two phases. The upper phase (containing proline) was measured with an ultraviolet spectrophotometer (Spectronic, Genesys2, USA) at 520 nm using toluene as the blank. The proline concentration was quantified based on a standard curve using D-proline.
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To evaluate the drought stress tolerance of SOR plants, 1-month-old NT and SOR plants were subjected to water deficit for 7 days. Before withholding the water supply, the plants were irrigated with similar quantities of water for 1 week; no obvious differences were observed between SOR and NT plants. After 4 days of withholding water, we observed severe wilting of the NT plants, while the SOR plants exhibited less wilting. When the plants were re-watered after drought stress treatment, all SOR lines recovered successfully, with only a few withered leaves, whereas NT plants were almost dead and failed to recover from dehydration conditions (Fig 4A). IbOr transcript levels were significantly higher in SOR plants than in NT plants after drought treatment (Fig 4B). To assess the degree of drought stress, the RWC values, a good indicator of the turgidity maintained by plants, were estimated using leaves from alfalfa plants before and after withholding water for 3 days. After 3 days of drought stress treatment, severe water loss was observed in the leaves of NT plants, with only 61.2% water content, while SOR2, SOR3, and SOR8 plants maintained significantly higher RWCs: 92.1%, 93.6%, and 94.3%, respectively (Fig 4C). In addition, SOR plants exhibited significantly lower MDA levels than NT plants after 3 days of drought stress treatment (Fig 4D). The degree of cell membrane damage under drought stress was greater in NT plants than in SOR plants. Under control conditions, the levels of free proline were similar between NT and SOR plants. However, higher levels of free proline were detected in the three SOR lines than in NT plants after 3 days of drought stress treatment (Fig 4E). Moreover, the SOR lines did not exhibited distinct color changes in stress-treated leaves following DAB staining, whereas a dark brown color through the leaf veins was observed in NT plants due to high levels of H2O2 under drought stress conditions (Fig 4F). These results indicate that the presence of IbOr increases drought stress tolerance in SOR plants.
In plants under salt and drought stress, increased ROS production results in the accumulation of MDA due to membrane lipid peroxidation . Stress-induced membrane damage and cell membrane stability are efficient criteria used to assess the degree of stress tolerance in plants . Our results show that transgenic alfalfa plants expressing IbOr exhibited less cellular membrane injury, with markedly lower MDA contents, than NT plants (Figs 3D and 4D). Simultaneously, plants often accumulate compatible osmoprotectants, such as free proline, which protect them from stress [39, 40]. In the current study, SOR alfalfa plants under salt and drought stress exhibited a distinct increase in proline accumulation compared with NT plants (Figs 3E and 4E); this increase might contribute to the increased salt and drought tolerance of transgenic alfalfa. Increase of proline contents may be just because NT is dying and then produce less proline than transgenic lines. Despite their deep root system, the growth of most alfalfa cultivars is limited by drought stress . We found that IbOr increased the viability of SOR plants under stress conditions, suggesting that this gene will be useful for improving plant tolerance to multiple abiotic stresses. 350c69d7ab