Plant materials

The seeds of chilling-sensitive maize (Zea mays L.) inbred line Mo17 (Zheng et al., 2006) were used in this study. Dry seeds were imbibed between wet filter papers in a growth chamber at 25 °C, and sampled at 0, 3, 6, 9, 12, 24, and 36 h after imbibition (hai) until 50% radicle protrusion. The fresh weights of samples were recorded to determine the time-course of water absorption. Several critical points during seed germination were determined as 9, 24, 32, and 54 hai. Then, dry seeds were respectively subjected to 25 and 15 °C with 12 h/12 h light–dark cycles, and sampled at 0, 9, 24, 32, and 54 hai for electrolyte leakage tests and analysis of annexin expression.

Because the membrane damage mainly occurred at the early stage of seed imbibition, seeds were imbibed in 5 °C water for 2 h and then kept at 25 °C for 7 h for recovery (5–25 °C). Sampling time points were set as 0, 0.5, 2, 3, 6, and 9 hai to further determine the membrane injury of maize embryos and expression levels of ZmANN33 and ZmANN35. For the purpose of studying the effect of calcium ions on annexin expression, 5 mM EGTA (a Ca²⁺ chelator) solutions were used for contrastive analyses.

Embryos of all samples were collected rapidly, frozen in liquid nitrogen, and stored at −80 °C for subsequent analysis.