Localization and expression of CRSH transcript, level of calcium ions, and cell cycle activity during Brassica napus L. seed development

Seeds are used as human food and animal feed, but the seed is also the onset of a new life. Therefore, seeds are a crucial agricultural product for global food security. Among several mechanisms involved in seed formation and maturation, the stringent response seems to be of great but rather overlooked significance. Plant homologs of bacterial RelA/SpoT proteins, called RSH, metabolize hyperphosphorylated regulatory nucleotides guanosine tetra- and pentaphosphate ((p)ppGpp, alarmones) which are involved in varied aspects of plant growth and development. The role of plant RSHs and alarmones in seed development remains elusive. In this study, the possible function of Ca2+-dependent RSH (CRSH) proteins during seed formation and maturation was verified. In silico analysis of plant CRSH proteins showed that EF-hand calcium-binding motif was highly conserved in monocotyledonous and dicotyledonous plants. Remarkably, this motif is present only in plant CRSH and has not been found in bacterial homologs. In developing Brassica napus (canola) seeds we analyzed the level and the localization of BnCRSH mRNAs by RT-qPCR and in situ localization, respectively. Further, we examined the cell cycle activity via flow cytometry, and the level of calcium ions using scanning electron microscopy. Our results showed that DNA replication intensity was at the highest level in seeds at the early stages of development, and then constantly decreased to the minimal level reached 70 days after flowering. In contrast, the level of calcium ions and BnCRSH transcript increased during canola seed maturation. The results of this study strongly suggest that calcium-dependent stringent response plays a significant role during canola seed development.