5-azacytidine affects triticale microspore embryogenesis and is associated with modulation of reactive oxygen...

In theory, microspore embryogenesis (ME) is the most efficient method for producing doubled haploids, however the actual effectiveness of this process is strongly genotype dependent. Here, we analysed how treatment with 5 µM 5-azacytidine (AC) is associated with early physiological and molecular events in triticale anthers and microspores redirected towards embryogenic development. The effectiveness of ME was evaluated in anther cultures of two DH lines with contrasting ME responsiveness and was related to differences in reactive oxygen species (ROS) generation, cysteine protease activity profiling, and the frequency of programmed cell death (PCD).

AC increased microspores viability, doubled the number of embryo-like structures (ELS), and improved green plant regeneration in the responsive DH28 line, but did not efficiently overcome the recalcitrancy of the DH19 line. AC treatment elevated total ROS level and reduced DNA fragmentation in DH28. However, the accumulation of one of the ROS, hydrogen peroxide (H₂O₂), was not affected by AC treatment but significantly higher compared with the recalcitrant DH19. Activity-based protease profiling using specific inhibitors revealed that AC treatment strongly suppress of PCD-associated cysteine proteases, particularly papain-like cysteine proteases (PLCPs) and vacuolar processing enzymes (VPEs-like) in DH28. On the contrary, caspase-3-like activity was consistently present and appeared to play a regulatory or signalling role rather than functioning as a primary executor of PCD. In contrast, DH19 maintained higher overall protease activity and showed no AC-related changes in ROS generation.

AC enhances the effectiveness of triticale ME in a genotype-dependent manner, and its positive effect is accompanied with reduced PCD-associated proteolysis during early cellular reprogramming. Genotypic differences in the ability to suppress PCD-associated protease activity may therefore contribute to variation in ME efficiency. The observed relationships between AC treatment, ROS levels and protease activity remain correlative and warrant future mechanistic investigation to clarify the causal links among these processes.