Abstract
Our findings provide new insights into the molecular mechanisms that regulate N metabolism in barley and potentially other cereal crops, offering valuable perspectives for enhancing N use efficiency in agricultural practices. Efficient nitrogen (N) utilization is essential for plant growth, especially during seed development and germination. In barley (Hordeum vulgare), the asparagine synthetase gene HvASN1 is essential for nitrogen transport and storage, synthesizing asparagine, a key molecule in N recycling. The phylogenetic analysis indicates that HvASN1 clusters with Arabidopsis AtASN1 and shares high similarity with HvASN2, suggesting a conserved role in N metabolism. A detailed characterization of a ~ 500 bp HvASN1 promoter region revealed a conserved GCN-like cis-element. Transient expression assays in Nicotiana benthamiana demonstrated that the wild-type promoter significantly increases luciferase activity under dark conditions, whereas mutation of the GCN-like element reduces this activity, highlighting its role in light-responsive gene regulation. Further investigation identified the bZIP transcription factor HvbZIP53 as an activator of the HvASN1 promoter through binding to the GCN-like element. This activation is finely tuned by sucrose via a conserved upstream open reading frame (uORF) in HvbZIP53's 5' UTR, which mediates sucrose-induced repression of translation. Additionally, yeast two-hybrid assays and transient expression studies in Arabidopsis provided evidence that HvbZIP53 physically interacts with HvBLZ1, a C group bZIP factor, resulting in a synergistic enhancement of HvASN1 expression. The spatial and temporal expression analyses further revealed that HvASN1, HvbZIP53, and HvBLZ1 are co-expressed in key seed tissues during development and germination. These findings indicate a complex regulatory network integrating environmental and metabolic signals to modulate N metabolism in barley, with implications for improving N use efficiency in cereal crops.