Redirecting vacuolar nitrate transport improves nitrogen use efficiency and seed protein content

Improving seed protein content without compromising carbon allocation or yield is a major challenge for enhancing nitrogen use efficiency. Here, we show that redirecting vacuolar nitrate transport through concurrent manipulation of tonoplast proteins controlling nitrate storage or export provides an effective lever to reprogram nitrogen allocation from leaves toward the seeds. Using Arabidopsis thaliana Ws lines disrupted for the vacuolar CLC-a nitrate importer and/or overexpressing the NRT2.7 tonoplast nitrate exporter, we show that plants combining the two modifications (35S::NRT2.7(clc-a)) integrate reduced nitrogen retention in vegetative tissues with increased nitrogen allocation to seeds. As a result, 35S::NRT2.7(clc-a) plants exhibit the strongest increase in seed protein content among all genotypes (approximately +25%) without affecting seed yield, carbon concentration, or lipid composition. Altered vacuolar nitrate fluxes in 35S::NRT2.7(clc-a) stimulate nitrate assimilation, enhance nitrate reductase activity and amino acid biosynthetic pathways, and drive coordinated reprogramming of nitrogen and carbon metabolisms. Through 15N pulse chase experiments, we confirmed that 35S::NRT2.7(clc-a) shows the highest nitrogen remobilization efficiency toward seeds. Overexpression of the barley NRT2.7 homolog HvNRT2.10 in Arabidopsis wild type and clc-a backgrounds reproduces the key features of 35S::NRT2.7 phenotype, demonstrating the conservation of NRT2.7 regulatory effects on plant metabolism across species. Together, these findings identify vacuolar nitrate transport as a promising target to modulate grain protein content in cereals through genetic strategies acting on nitrogen storage and remobilization.