4.4. Stress Tolerance Assays and Measurements of Physiological Indices

In preparation for the germination assays, ~100 seeds were surface-sterilized and sown on MS medium supplemented with different concentrations of NaCl (100 mM and 150 mM), mannitol (250 mM and 300 mM), CuSO₄ (50 uM and 100 uM), CdSO₄ (50 uM and 100 uM), ETH (400 uM), JA (200 uM), and ABA (1.0 uM). Seeds were vernalized at 4 °C for 3 days before growing in a growth chamber. The root length and fresh weight were measured on day 7 after growing.

For the plant growth assays, 7-day-old OE-GmbZIP152 and WT seedlings were transferred into the compost soil. We treated three-week-old WT and OE-GmbZIP152 plants with NaCl (100 mM and 150 mM), mannitol (250 mM and 300 mM), CuSO₄ (50 uM and 100 uM), and CdSO₄ (50 uM and 100 uM) for 18 days and measured the plant height. All experiments were repeated three times.

To explore the expression profile of GmbZIP152, two-week-old soybean seedling leaves were infected with S. sclerotiorum and seedlings were treated with 150 mM NaCl for salt conditions, 400 mM mannitol for drought conditions, 150 uM CuSO₄, and 150 uM CdSO₄ for heavy metal conditions, 400 uM ETH, 150 uM JA, 1.0 uM ABA, and 250 uM SA. In addition, the eight-week-old mature soybean was treated with NaCl, mannitol, CuSO₄, CdSO₄, ETH, JA, ABA, and SA. The leaves detected the expression level of GmbZIP152.

The three-week-old plants were treated with SOD, CAT, and POD Activity Detection kit (Solarbio, Beijing, China) for 24 h to measure the physiological indices, according to the manufacturer’s instructions.