Quantitative Proteomics Reveals the Protein Regulatory Network of <i>Anabaena</i> sp. PCC 7120 under Nitrogen Deficiency

Anabaena sp. PCC 7120 (Anabaena 7120) is a photoautotrophic filamentous cyanobacterium capable of fixing atmospheric nitrogen. It is a model organism used for studying cell differentiation and nitrogen fixation. Under nitrogen deficiency, Anabaena 7120 forms specialized heterocysts capable of nitrogen fixation. However, the molecular mechanisms involved in the cyanobacterial adaptation to nitrogen deficiency are not well understood. Here, we employed a label-free quantitative proteomic strategy to systematically investigate the nitrogen deficiency response of Anabaena 7120 at different time points. In total, 363, 603, and 669 proteins showed significant changes in protein abundance under nitrogen deficiency for 3, 12, and 24 h, respectively. With mapping onto metabolic pathways, we revealed proteomic perturbation and regulation of carbon and nitrogen metabolism in response to nitrogen deficiency. Functional analysis confirmed the involvement of nitrogen stress-responsive proteins in biological processes, including nitrogen fixation, photosynthesis, energy and carbon metabolism, and heterocyst development. The expression of 10 proteins at different time points was further validated by using multiple reaction monitoring assays. In particular, many dysregulated proteins were found to be time-specific and involved in heterocyst development, providing new candidates for future functional studies in this model cyanobacterium. These results provide novel insights into the molecular mechanisms of nitrogen stress responses and heterocyst development in Anabaena 7120.鱼腥藻属PCC 7120菌株（Anabaena sp. PCC 7120，简称Anabaena 7120）是一类可固定大气氮的光合自养型丝状蓝细菌，亦是用于研究细胞分化与固氮机制的经典模式生物。在氮缺乏胁迫条件下，Anabaena 7120会形成具备固氮功能的特化异形胞（heterocysts）。然而，目前学界对蓝细菌适应氮缺乏胁迫的分子调控机制仍缺乏清晰认知。本研究采用无标记定量蛋白质组学策略（label-free quantitative proteomic strategy），系统探究了Anabaena 7120在氮缺乏处理不同时间节点后的蛋白质组应答特征。结果显示，在氮缺乏处理3、12、24小时后，分别有363、603和669种蛋白质的丰度发生显著改变。通过将差异蛋白映射至代谢通路，我们揭示了碳、氮代谢在氮缺乏响应过程中的蛋白质组扰动与调控模式。功能分析证实，氮胁迫响应蛋白参与了固氮、光合作用、能量与碳代谢以及异形胞发育等多个生物学过程。我们进一步通过多反应监测实验（multiple reaction monitoring assays）验证了10种蛋白质在不同时间节点的表达水平。尤为重要的是，大量差异表达蛋白呈现时间特异性特征，并参与异形胞发育过程，为该模式蓝细菌的后续功能研究提供了全新的候选靶点。本研究结果为解析Anabaena 7120的氮胁迫响应与异形胞发育的分子机制提供了全新的研究视角。