Abstract
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.
Highlights
Life in all three domains is sustained by membrane protein complexes participating in circuits that couple redox reactions and proton pumping, with the generation of ATP and NAD(P)H
In attempts to differentiate rates of O2 reduction between the acceptor side of PSI and the reduced PQ pool/cytochromeReexamining the Efficacy of DNP-INT (Cyt)-b6f complex, we have tested the efficacy of DBMIB and dinitrophenylether of iodonitrothymol (DNP-INT), two well characterized chemicals used to truncate PSI from the photosynthetic electron transfer chain (PETC), to validate that PSI was not reduced during our measurements
In a clear demonstration of successful PSI truncation from the rest of the PETC, the level of P700+ was largely unaffected following the single turnover (ST) and multiple turnover (MT) actinic pulses in both leaf and thylakoid samples treated with 10 μM DCMU (Blue lines) and 10 μM DBMIB (Pink lines)
Summary
Life in all three domains is sustained by membrane protein complexes participating in circuits that couple redox reactions and proton pumping, with the generation of ATP and NAD(P)H. The results from O2 electrode measurements with isolated thylakoids were interpreted as the ability of DNP-INT to allow semiquinone formation, whilst simultaneously blocking Cyt-f reduction (Mubarakshina and Ivanov, 2010), which made DNPINT a critical tool in truncating the PETC at Cyt-b6f It has resulted in the wide use of DNP-INT as an alternative to DBMIB for truncation of PSI from the PETC, in works to explore reactive oxygen species (ROS) formation and scavenging pathways involving both reduced PQ-pool and semiquinones (Khorobrykh and Ivanov, 2002; Heyno et al, 2009; Mubarakshina and Ivanov, 2010; Borisova-Mubarakshina et al, 2018). MIMS was used to distinguish the O2 consuming and O2 producing reactions occurring simultaneously in isolated thylakoid samples to test conclusions drawn from O2 electrode data, and was used to measure rates of CO2 fixation in leaf discs infiltrated with DNP-INT
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