Regenerative potential, metabolic profile, and genetic stability of Brachypodium distachyon embryogenic calli as affected by successive subcultures

Brachypodium distachyon, a model species for forage grasses and cereal crops, has been used in studies seeking improved biomass production and increased crop yield for biofuel production purposes. Somatic embryogenesis (SE) is the morphogenetic pathway that supports in vitro regeneration of such species. However, there are gaps in terms of studies on the metabolic profile and genetic stability along successive subcultures. The physiological variables and the metabolic profile of embryogenic callus (EC) and embryogenic structures (ES) from successive subcultures (30, 60, 90, 120, 150, 180, 210, 240, and 360-day-old subcultures) were analyzed. Canonical discriminant analysis separated EC into three groups: 60, 90, and 120 to 240 days. EC with 60 and 90 days showed the highest regenerative potential. EC grown for 90 days and submitted to SE induction in 2 mg L−1 of kinetin-supplemented medium was the highest ES producer. The metabolite profiles of non-embryogenic callus (NEC), EC, and ES submitted to principal component analysis (PCA) separated into two groups: 30 to 240- and 360-day-old calli. The most abundant metabolites for these groups were malonic acid, tryptophan, asparagine, and erythrose. PCA of ES also separated ages into groups and ranked 60- and 90-day-old calli as the best for use due to their high levels of various metabolites. The key metabolites that distinguished the ES groups were galactinol, oxaloacetate, tryptophan, and valine. In addition, significant secondary metabolites (e.g., caffeoylquinic, cinnamic, and ferulic acids) were important in the EC phase. Ferulic, cinnamic, and phenylacetic acids marked the decreases in the regenerative capacity of ES in B. distachyon. Decreased accumulations of the amino acids aspartic acid, asparagine, tryptophan, and glycine characterized NEC, suggesting that these metabolites are indispensable for the embryogenic competence in B. distachyon. The genetic stability of the regenerated plants was evaluated by flow cytometry, showing that ploidy instability in regenerated plants from B. distachyon calli is not correlated with callus age. Taken together, our data indicated that the loss of regenerative capacity in B. distachyon EC occurs after 120 days of subcultures, demonstrating that the use of EC can be extended to 90 days

Oxygen Perception in Plants

In aerobic organisms oxygen is a rate-limiting substrate for the efficient production of energy, and therefore they need to adjust their metabolism to the availability of oxygen. For this reason, eukaryotes and prokaryotes independently developed mechanisms to perceive oxygen availability and integrate this into developmental and growth programs. Despite their ability to produce oxygen in the presence of light, plants can experience low oxygen conditions when the oxygen diffusion from the environment cannot satisfy the demand set by metabolic rates. The oxygen-sensing mechanism recently identified in plants shares striking similarities with those previously described in animal cells. While in bacteria the different oxygen-sensing pathways reported involve protein dimerization and phosphorylation cascades, in plants and animals this function is mediated by oxygendependent proteolysis. The plant oxygen-sensing pathway is regulated via the oxygen-dependent branch of the N-end rule, which regulates the stability of the group VII of the Ethylene Response Factors, key activators of the anaerobic response. Additionally, constitutively expressed ERF-VII proteins, such as RAP2.12, are bound to the acyl-CoA-binding proteins (ACBPs) at the plasma membrane and protected from aerobic degradation. In hypoxia, RAP2.12 is released from the membrane and relocalizes into the nucleus, where it activates the molecular response to oxygen deficiency. Additional factors, indirectly affected by oxygen availability, have also been suggested to play roles in the fine tuning of oxygen sensing in plants