Programmed Cell Death in the Endosperm Is a Hallmark of Seed Germination in Viola

Programmed cell death (PCD) is a pivotal biological process that occurs at various stages of plant development, including embryogenesis and seed germination. This study investigated whether the absence of PCD in endosperm cells is connected to the poor germination of Viola odorata seeds. Seeds of poorly germinating V. odorata and well-germinating V. × wittrockiana were either cold-stratified for 10 days or left untreated. Germination tests, tetrazolium viability tests, Western blot analyses for caspase-like proteases, and Terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) assays for DNA strand break detection were performed. The results revealed that V. odorata seeds did not germinate, regardless of stratification or lack thereof, whereas in V. × wittrockiana, stratification significantly increased their germination capacity (34 ± 6.5% vs. 56.5 ± 9.8% in non-stratified and stratified seeds, respectively). The tetrazolium viability test revealed that V. odorata seeds were nonviable (100% nonviable endosperm and 96% nonviable embryos in total), whereas the seeds of V. × wittrockiana were highly viable (63% and 59% endosperm and embryos in total, respectively). Western blot analysis revealed that in the germinating seeds of V. × wittrockiana, caspase-like activity was detected in the endosperm but not in the embryos, whereas in seeds that failed to germinate, the PCD signal in the endosperm was very weak. In the seeds of V. odorata, caspase-like activity was detected in the embryos and endosperm collected directly after 10 days of stratification, but no signal was detected in the seeds left to germinate for one month after cold stratification. TUNEL assays revealed DNA strand breaks in the peripheral part of the endosperm in V. odorata and in non-germinating V. × wittrockiana, whereas in the germinating seeds of V. × wittrockiana, DNA strand breaks were detected in the endosperm cells adjacent to the embryo. These findings indicate that endosperm-localized PCD facilitates nutrient mobilization to the embryo and seems crucial for successful germination. Overall, these results suggest that PCD contributes to the regulation of seed germination in Viola spp.