Table 1.
Gene-specific primers used for genomic and RT-PCR analysis.
Fig 1.
Development of transgenic alfalfa expressing IbOr under the control of the SWPA2 promoter (SOR plants).
(A) Diagram of the oxidative stress-inducible SWPA2 promoter: IbOr construct used for alfalfa transformation. Vertical bar shows the primer set (A2pro::IbOr) used for genomic PCR analysis. (B) Genomic DNA PCR analysis using the A2pro::IbOr primer set. Numbers (1–11) represent independent transgenic lines. M, size markers; NT, non-transgenic plant; PC, positive control. (C) RT-PCR analysis of 11 lines expressing stable IbOr gene integration in transgenic plants following 2 h of 5 μM MV treatment.
Fig 2.
Effects of MV-mediated oxidative stress on leaves of SOR and NT plants.
(A) Visible damage in leaves after 5 μM MV treatment for 24 h. (B) Ion leakage was measured after 0, 12, 24, and 36 h of MV treatment. Percentage of relative membrane permeability was calculated using 100% to represent the values obtained after autoclaving. (C) IbOr transcript levels after 12 h of 5 μM MV treatment. The expression levels of IbOr were normalized to that of the alfalfa Actin gene as the internal control. Data are expressed as the mean ± SD of three independent biological replicates. Bars labeled with asterisks show significant differences from that of NT at * P < 0.05 or ** P < 0.01 by t-test.
Fig 3.
Enhanced tolerance to high salinity in SOR plants.
(A) Plant growth under normal conditions (upper panel) and salt stress (250 mM NaCl) for 4 days (middle panel) and 7 days (lower panel). (B) Relative transcript levels of IbOr in leaves. (C) Relative chlorophyll contents of alfalfa plants after 3 days of salt treatment. (D) MDA contents in leaves after 3 days of salt treatment. (E) Proline contents of alfalfa plants after 3 days of salt treatment. (F) DAB staining for H2O2 accumulation in the third detached leaves after 3 days of 250 mM NaCl treatment. The values represent the mean ± SD of three independent replicates. Asterisks indicate a significant difference from that of NT at * P < 0.05 or ** P < 0.01 by t-test.
Fig 4.
Enhanced tolerance to drought stress in SOR plants.
(A) Phenotypes of 1-month-old NT and SOR plants before treatment (upper panel), after withholding water for 4 days (middle panel), and recovered phenotype after re-watering for 7 days (lower panel). (B) Transcript levels of IbOr after withholding water for 2 days. (C) RWC (%) in leaves after 3 days of water withholding. (D) MDA contents in leaves after 3 days of drought treatment. (E) Proline contents in leaves after 3 days of drought treatment. (F) DAB staining for H2O2 accumulation in the third detached leaves of alfalfa plants after 3 days of drought treatment. Values represent the mean ± SD of three independent replicates. Asterisks indicate a significant difference from that of NT at * P < 0.05 or ** P < 0.01 by t-test.
Fig 5.
Quantitative HPLC analysis of total carotenoid contents and carotenoid compounds in NT and SOR plants.
All levels are expressed as the mean (average content in grams dry weight) ± SD of two independent determinations with four biological repeats. Asterisks indicate a significant difference from that of NT at * P < 0.05 or ** P < 0.01 by t-test.
Fig 6.
Transcript analysis of carotenoid biosynthetic pathway genes in NT and SOR plants under drought stress.
Leaves (from same position) of plants treated with drought stress for 2 days were utilized. The expression level of each gene was normalized to that of the Actin gene of alfalfa as the internal control. MsPSY, phytoene synthase; MsCHY-β, β-carotene hydroxylase; MsLCY-β, lycopene β-cyclase; MsNCED, 9-cis-epoxycarotenoid dioxygenase. The values represent the mean ± SD of three independent biological replicates. Asterisks indicate a significant difference from that of NT at * P < 0.05 or ** P < 0.01 by t-test.