Cross-species transcriptomic plasticity and root architectural adaptations in Brachypodium (Brachypodium distachyon) and barley (Hordeum vulgare) genotypes under different ammonium and nitrate availability.

Nitrogen (N) form and availability shape the root phenotype by altering the root transcriptome and modulating root system architecture. However, it remains unclear whether these N-responsive architectural traits and their underlying regulatory networks are conserved or divergent between closely related grass species. Here, we compared root architectural and transcriptomic responses to nitrate and ammonium supply in Brachypodium distachyon and eight barley (Hordeum vulgare) genotypes under N starvation, optimal N supply, and high-N conditions. High-resolution root phenotyping revealed contrasting species-specific responses to N form. Barley generally showed enhanced lateral root elongation under nitrate but reduced total root length and limited lateral root proliferation under ammonium, indicating ammonium sensitivity. In contrast, B. distachyon maintained greater root architectural plasticity under ammonium, producing a highly branched and thinner root system. Cross-species module preservation and gene regulatory network analyses identified conserved hub genes, including FERONIA, peroxidase-like genes, CuAO-like genes, aspartyl proteases, and PEPC1-associated modules, as potential integrators of N signaling and root architectural remodeling in barley and B. distachyon. Taken together, our findings indicate that the two Pooideae species share conserved regulatory components but deploy them within distinct network contexts, leading to divergent root strategies in response to nitrate and ammonium. Ultimately, these results provide candidate genes and regulatory modules for improving root plasticity and N use efficiency in cereals.