Data_Sheet_1_Carbon and nitrogen metabolic regulation in freshwater plant Ottelia alismoides in response to carbon limitation: A metabolite perspective.docx

Carbon and nitrogen metabolism are basic, but pivotal metabolic pathways in plants and are tightly coupled. Maintaining the balance of carbon and nitrogen metabolism is critical for plant survival. Comprehensively revealing the metabolic balance of carbon–nitrogen interactions is important and helpful for understanding the adaptation of freshwater plants to CO2 limited aqueous environment. A comprehensive metabolomics analysis combined with physiological measurement was performed in the freshwater plant Ottelia alismoides acclimated to high and low CO2, respectively, for a better understanding of how the carbon and nitrogen metabolic adjustment in freshwater plants respond to carbon limitation. The present results showed that low CO2 acclimated O. alismoides exhibited significant diurnal titratable acidity and malate fluctuations, as well as an opposite diel pattern of starch change and high enzymatic activities required for crassulacean acid metabolism (CAM) photosynthesis, which indicates that CAM was induced under low CO2. Moreover, the metabolomic analysis showed that most intermediates of glycolysis, pentose phosphate pathway (PPP) and tricarboxylic acid (TCA) cycle, were increased under low CO2, indicative of active respiration in low-CO2-treated O. alismoides. Meanwhile, the majority of amino acids involved in pathways of glutamate and arginine metabolism, aspartate metabolism, and the branched-chain amino acids (BCAAs) metabolism were significantly increased under low CO2. Notably, γ-aminobutyric acid (GABA) level was significantly higher in low CO2 conditions, indicating a typical response with GABA shunt compensated for energy deprivation at low CO2. Taken together, we conclude that in low-CO2-stressed O. alismoides, CAM photosynthesis was induced, leading to higher carbon and nitrogen as well as energy requirements. Correspondingly, the respiration was greatly fueled via numerous starch degradation to ensure CO2 fixation in dark, while accompanied by linked promoted N metabolism, presumably to produce energy and alternative carbon sources and nitrogenous substances for supporting the operation of CAM and enhancing tolerance for carbon limitation. This study not only helps to elucidate the regulating interaction between C and N metabolism to adapt to different CO2 but also provides novel insights into the effects of CO2 variation on the metabolic profiling of O. alismoides.

碳氮代谢是植物体内基础且关键的代谢通路，二者紧密耦联。维持碳氮代谢平衡对植物存活至关重要。全面解析碳氮互作的代谢平衡，有助于理解淡水植物如何适应CO₂受限的水生环境。为深入解析淡水植物的碳氮代谢调控如何响应碳限制胁迫，本研究以分别适应高、低CO₂环境的淡水植物水车前（Ottelia alismoides）为材料，开展了全面的代谢组学（metabolomics）分析结合生理指标测定。本研究结果显示，经低CO₂驯化的水车前表现出显著的昼夜可滴定酸度与苹果酸含量波动，同时淀粉含量变化呈现相反的昼夜节律模式，且具备景天酸代谢（crassulacean acid metabolism, CAM）光合作用所需的高酶活性，表明低CO₂环境下诱导了CAM途径。此外，代谢组学分析显示，低CO₂条件下糖酵解、磷酸戊糖途径（pentose phosphate pathway, PPP）及三羧酸循环（tricarboxylic acid, TCA cycle）的多数中间产物含量上调，表明低CO₂处理的水车前呼吸作用更为活跃。与此同时，低CO₂条件下参与谷氨酸代谢、精氨酸代谢、天冬氨酸代谢及支链氨基酸（branched-chain amino acids, BCAAs）代谢途径的多数氨基酸含量显著上调。值得注意的是，低CO₂环境下γ-氨基丁酸（γ-aminobutyric acid, GABA）的含量显著升高，表明GABA分流（GABA shunt）作为典型响应机制，弥补了低CO₂条件下的能量匮乏。综上，本研究认为在低CO₂胁迫的水车前中，CAM光合作用被诱导，进而引发更高的碳、氮及能量需求。相应地，植物通过大量淀粉降解为暗反应中的CO₂固定提供底物支撑，大幅激活呼吸作用；同时伴随氮代谢的协同上调，推测其旨在生成能量、替代性碳源及含氮物质，以维持CAM途径的运行并增强对碳限制的耐受性。本研究不仅阐明了碳氮代谢的调控互作如何适应不同CO₂环境，还为理解CO₂浓度变化对水车前代谢谱的影响提供了全新视角。