Data_Sheet_4_A Commonly Used Photosynthetic Inhibitor Fails to Block Electron Flow to Photosystem I in Intact Systems.PDF

In plant science, 2,4-dinitrophenylether of iodonitrothymol (DNP-INT) is frequently used as an alternative to 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone (DBMIB) to examine the capacity of plastoquinol and semiquinone to reduce O2. DNP-INT is considered to be an effective inhibitor of the photosynthetic electron transfer chain (PETC) through its binding at the Q0 site of Cyt-b6f. The binding and action of DNP-INT has been previously characterized spectroscopically in purified Cyt-b6f complex reconstituted with Plastocyanin, PSII membranes and plastoquinone, as well as in isolated thylakoids based on its property to block MV-mediated O2 consumption. Contrary to the conclusions obtained from these experiments, we observed clear reduction of P700+ in samples incubated with DNP-INT during our recent investigation into the sites of oxygen consumption in isolated thylakoids. Therefore, we carried out an extensive investigation of DNP-INT’s chemical efficacy in isolated thylakoids and intact leaves. This included examination of its capacity to block the PETC before PSI, and therefore its inhibition of CO2 fixation. P700 redox kinetics were measured using Dual-PAM whilst Membrane Inlet Mass Spectrometry (MIMS) was used for simultaneous determination of the rates of O2 evolution and O2 consumption in isolated thylakoids and CO2 fixation in intact leaves, using two stable isotopes of oxygen (16O2, 18O2) and CO2 (12C, 13C), respectively. Based on these investigations we confirmed that DNP-INT is unable to completely block the PETC and CO2 fixation, therefore its use may produce artifacts if applied to isolated thylakoids or intact cells, especially when determining the locations of reactive oxygen species formation in the photosynthetic apparatus.

在植物科学研究中，碘硝基胸腺醇2,4-二硝基苯醚（2,4-dinitrophenylether of iodonitrothymol，DNP-INT）常被用作2,5-二溴-6-异丙基-3-甲基-1,4-苯醌（2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone，DBMIB）的替代试剂，用于探究质体醌醇（plastoquinol）与半醌（semiquinone）还原氧气的能力。DNP-INT被认为是一类高效的光合电子传递链（photosynthetic electron transfer chain，PETC）抑制剂，其作用靶点为细胞色素b₆f（Cyt-b₆f）的Q₀位点。此前已有研究基于DNP-INT可阻断MV介导的氧气消耗这一特性，在重组了质体蓝素（Plastocyanin）、光系统II（PSII）膜与质体醌的纯化Cyt-b₆f复合物中，以及在分离的类囊体（thylakoids）中，通过光谱学手段对DNP-INT的结合方式与作用模式进行了表征。但与上述实验所得结论相悖的是，我们在近期针对分离类囊体氧气消耗位点的研究中发现，经DNP-INT孵育的样品中氧化态P700（P700+）发生了显著还原。为此，我们针对DNP-INT在分离类囊体与完整叶片中的化学效应开展了系统性研究，内容涵盖其阻断光系统I（PSI）上游光合电子传递链的能力，以及由此对二氧化碳固定产生的抑制作用。研究采用双通道脉冲振幅调制荧光仪（Dual-PAM）测定P700的氧化还原动力学；同时利用膜进样质谱法（Membrane Inlet Mass Spectrometry，MIMS），分别借助氧的两种稳定同位素（¹⁶O₂、¹⁸O₂）与碳的两种稳定同位素（¹²C、¹³C），同步检测分离类囊体的氧气释放与消耗速率，以及完整叶片的二氧化碳固定速率。基于上述研究，我们证实DNP-INT无法完全阻断光合电子传递链与二氧化碳固定过程。因此，若将其应用于分离类囊体或完整细胞的相关实验，可能会引入人为假象，尤其是在光合装置中活性氧（reactive oxygen species）生成位点的检测工作中。