Abstract
Rcd1 (radical-induced cell death1) is an Arabidopsis thaliana mutant, which exhibits high tolerance to paraquat [methyl viologen (MV)], herbicide that interrupts photosynthetic electron transport chain causing the formation of superoxide and inhibiting NADPH production in the chloroplast. To understand the biochemical mechanisms of MV-resistance and the role of RCD1 in oxidative stress responses, we performed metabolite profiling of wild type (Col-0) and rcd1 plants in light, after MV exposure and after prolonged darkness. The function of RCD1 has been extensively studied at transcriptomic and biochemical level, but comprehensive metabolite profiling of rcd1 mutant has not been conducted until now. The mutant plants exhibited very different metabolic features from the wild type under light conditions implying enhanced glycolytic activity, altered nitrogen and nucleotide metabolism. In light conditions, superoxide production was elevated in rcd1, but no metabolic markers of oxidative stress were detected. Elevated senescence-associated metabolite marker levels in rcd1 at early developmental stage were in line with its early-senescing phenotype and possible mitochondrial dysfunction. After MV exposure, a marked decline in the levels of glycolytic and TCA cycle intermediates in Col-0 suggested severe plastidic oxidative stress and inhibition of photosynthesis and respiration, whereas in rcd1 the results indicated sustained photosynthesis and respiration and induction of energy salvaging pathways. The accumulation of oxidative stress markers in both plant lines indicated that MV-resistance in rcd1 derived from the altered regulation of cellular metabolism and not from the restricted delivery of MV into the cells or chloroplasts. Considering the evidence from metabolomic, transcriptomic and biochemical studies, we propose that RCD1 has a negative effect on reductive metabolism and rerouting of the energy production pathways. Thus, the altered, highly active reductive metabolism, energy salvaging pathways and redox transfer between cellular compartments in rcd1 could be sufficient to avoid the negative effects of MV-induced toxicity.
Highlights
Reactive oxygen species (ROS) such as superoxide (O2-) and hydrogen peroxide (H2O2) are important signaling molecules inevitably formed in aerobic energy metabolism
The a-KG/ succinate ratio, which describes the a-ketoglutarate dehydrogenase (a-KGDH) status, decreased in Col-0, but again remained in the same level as in light conditions in rcd1. These results indicate that methyl viologen (MV) exposure does not have the same effect to the mitochondrial redox status in rcd1 as in wild-type Col-0, even though increased oxidative stress marker levels indicate ROS production in all cell compartments in both lines during MV exposure (Figures 4C, D)
We propose that overactive reductive metabolism, activation of energy salvaging pathways and efficient redox transfer between organelles is sufficient to overcome the negative effects of MV-induced chloroplastic oxidative stress in the mutant plants
Summary
Reactive oxygen species (ROS) such as superoxide (O2-) and hydrogen peroxide (H2O2) are important signaling molecules inevitably formed in aerobic energy metabolism. Basal levels of ROS are required for normal plant performance and development, in excess they cause oxidative stress that could damage cells and trigger physiological and programmed metabolic pathways, which can induce cell death (Noctor et al, 2015; Mittler, 2017; Waszczak et al, 2018). Exposure to excessive levels of ROS causes the down-regulation of anabolic metabolism (e.g. Calvin cycle) while favoring catabolic metabolism, such as oxidative pentose phosphate pathway (OPPP) and lipid, protein and starch degradation, to provide substrates for the production of ATP and reducing power in the form of NAD(P)H. In Arabidopsis, changes in gene expression and sugar levels indicated altered metabolism in response to treatment with ROS-generating herbicide, methyl viologen (MV), and the responses resembled transcriptomic changes in plants adapted to darkness (Scarpeci and Valle, 2008)
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