Auxin Metabolite Balance During Haploid and Zygotic Oat Embryo Development-Quantitative and Localization Studies

Auxins play a critical role in establishing the embryo axis and embryonic pattern. Our study aimed to determine the developmental stage of 21-day old oat (Avena sativa L.) haploid embryos, obtained by distant crossing with maize, and examined oat zygotic embryos at different developmental stages for their levels of endogenous indole-3-acetic acid (IAA), its metabolites, and IAA localization. The content of auxin metabolites was determined by HPLC-MS/MS, while IAA visualization in embryos was performed by immunohistochemistry and observed under confocal microscopy. We found that 21-day-old haploid embryos contained half the IAA concentration of age-matched zygotic embryos. Simultaneously, the total conjugated auxins (IAA-Asp, IAA-Glu, meIAA) were higher than in zygotic embryos, regardless of their age. Immunolocalization revealed IAA accumulation in embryos aligned with regions of tissue differentiation (e.g., shoot apical meristem, radicle primordium, and coleptile). We conclude that limited morphogenetic progression, evidenced by microscopic sections accompanied by changes in IAA content and distribution in haploid embryos, indicates a developmental stage earlier than the coleoptilar stage of zygotic embryos which occurs 9 days after pollination. Our findings may be useful in embryo rescue techniques, suggesting modulation of auxin concentration in in vitro culture.