Contreras Estefanía, Alonso Rosario, Pastor-Mora Elena, Ceballos Mar G, Vicente-Carbajosa Jesús, Iglesias-Fernández Raquel
Centro de Biotecnología y Genómica de Plantas-Severo Ochoa (CBGP, UPM-INIA), Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (CSIC/INIA), 28223, Pozuelo de Alarcón, Spain.
Departamento de Biotecnología-Biología Vegetal, Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de Biosistemas, UPM, 28040, Madrid, Spain.
Planta. 2025 Jun 9;262(1):20. doi: 10.1007/s00425-025-04730-0.
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.
我们的研究结果为调控大麦及其他潜在谷类作物氮代谢的分子机制提供了新见解，为提高农业生产中的氮利用效率提供了有价值的观点。高效的氮利用对植物生长至关重要，尤其是在种子发育和萌发期间。在大麦（Hordeum vulgare）中，天冬酰胺合成酶基因HvASN1对氮的运输和储存至关重要，它能合成天冬酰胺，这是氮循环中的关键分子。系统发育分析表明，HvASN1与拟南芥AtASN1聚类，且与HvASN2具有高度相似性，表明其在氮代谢中具有保守作用。对约500 bp的HvASN1启动子区域进行详细表征，发现了一个保守的类GCN顺式元件。在本氏烟草中进行的瞬时表达分析表明，野生型启动子在黑暗条件下显著提高荧光素酶活性，而类GCN元件的突变则降低了这种活性，突出了其在光响应基因调控中的作用。进一步研究确定bZIP转录因子HvbZIP53通过与类GCN元件结合而成为HvASN1启动子的激活因子。这种激活通过蔗糖在HvbZIP53的5'UTR中的保守上游开放阅读框（uORF）进行精细调节，该uORF介导蔗糖诱导的翻译抑制。此外，酵母双杂交试验和在拟南芥中的瞬时表达研究提供了证据，表明HvbZIP53与C组bZIP因子HvBLZ1发生物理相互作用，从而协同增强HvASN1的表达。时空表达分析进一步揭示，HvASN1、HvbZIP53和HvBLZ1在种子发育和萌发期间的关键种子组织中共同表达。这些发现表明存在一个复杂的调控网络，整合环境和代谢信号以调节大麦中的氮代谢，这对提高谷类作物的氮利用效率具有重要意义。
