Sena Florencia, Couture Camila, Berais-Rubio Andrés, Millar A Harvey, Signorelli Santiago
Food and Plant Biology Group, Departamento de Biología Vegetal, Facultad de Agronomía, Universidad de la República, Montevideo, Uruguay.
School of Molecular Sciences, The University of Western Australia, Crawley, WA 6009, Australia.
Plant Sci. 2025 Nov;360:112735. doi: 10.1016/j.plantsci.2025.112735. Epub 2025 Aug 26.
Photosynthesis and respiration are fundamental metabolic processes in plants, tightly connected through shared substrates, energy dynamics, and redox balance. Arabidopsis is the key genetic model for plants but monitoring these sorts of physiological processes presents significant challenges using traditional gas-exchange or fluorescence-based techniques due to the small size of intact Arabidopsis thaliana (arabidopsis) seedlings. Here, we validate and characterize the use of Clark-type oxygen electrodes, specifically the Hansatech Oxytherm+P system, to quantify both photosynthetic and respiratory activity in intact arabidopsis seedlings. By monitoring oxygen evolution in dark and light phases, we demonstrate that oxygen consumption and production correspond to mitochondrial respiration and photosynthesis, respectively. These processes were modulated by tissue biomass, light intensity, developmental stage, and stress conditions. Specific inhibitors such as potassium cyanide and DCMU confirmed that the recorded changes in oxygen concentrations reflected mitochondrial cytochrome oxidase activity and photosystem electron transport-dependent oxygen production, respectively. Moreover, oxygen evolution increased significantly with bicarbonate supplementation, validating the system's sensitivity to carbon fixation. We further showed that photosynthetic activity measured with this method correlates with a quantitative green index and responds dynamically to de-etiolation, abiotic stress (salt, osmotic, oxidative), and temperature shifts. Our study lays the groundwork for measuring photosynthesis based on oxygen evolution and respiration in arabidopsis knockout mutants, CRISPR lines, overexpression lines, and ecotypes using Clark-type oxygen electrodes and highlights key considerations and limitations to consider when applying this approach. This platform could also be adapted for many other small tissue plant samples.
光合作用和呼吸作用是植物的基本代谢过程，通过共享底物、能量动态和氧化还原平衡紧密相连。拟南芥是植物的关键遗传模型，但由于完整的拟南芥幼苗体积小，使用传统的气体交换或基于荧光的技术监测这类生理过程面临重大挑战。在此，我们验证并表征了Clark型氧电极（特别是Hansatech Oxytherm+P系统）在量化完整拟南芥幼苗光合和呼吸活性方面的应用。通过监测黑暗和光照阶段的氧气释放，我们证明了氧气消耗和产生分别对应于线粒体呼吸作用和光合作用。这些过程受到组织生物量、光照强度、发育阶段和胁迫条件的调节。特定抑制剂如氰化钾和敌草隆证实，记录的氧气浓度变化分别反映了线粒体细胞色素氧化酶活性和光系统电子传递依赖的氧气产生。此外，补充碳酸氢盐后氧气释放显著增加，验证了该系统对碳固定的敏感性。我们进一步表明，用这种方法测量的光合活性与定量绿色指数相关，并对去黄化、非生物胁迫（盐、渗透、氧化）和温度变化做出动态响应。我们的研究为使用Clark型氧电极测量拟南芥基因敲除突变体、CRISPR系、过表达系和生态型中的氧气释放光合作用和呼吸作用奠定了基础，并突出了应用该方法时需要考虑的关键因素和局限性。这个平台也可适用于许多其他小组织植物样本。
