Sensing and signaling of oxidative stress in chloroplasts by inactivation of the SAL1 phosphoadenosine phosphatase
Wednesday, 2016/08/03 | 08:13:51
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Kai Xun Chan, Peter D. Mabbitt, Su Yin Phua, Jonathan W. Mueller, Nazia Nisar, Tamara Gigolashvili, Elke Stroeher, Julia Grassl, Wiebke Arlt, Gonzalo M. Estavillo, Colin J. Jackson, and Barry J. Pogson SignificanceManagement of oxidative stress in plant chloroplasts involves signaling pathways to the nucleus that trigger stress response mechanisms. Yet, how oxidative stress is initially sensed in the chloroplast to activate accumulation of a stress signal remains enigmatic. We show that inactivation of a phosphatase, SAL1, by oxidative stress in chloroplasts controls accumulation of its substrate, as a plant stress signal. This regulatory mechanism is highly conserved across the plant kingdom and confers a second function to this metabolic enzyme as an oxidative stress sensor. AbstractIntracellular signaling during oxidative stress is complex, with organelle-to-nucleus retrograde communication pathways ill-defined or incomplete. Here we identify the 3′-phosphoadenosine 5′-phosphate (PAP) phosphatase SAL1 as a previously unidentified and conserved oxidative stress sensor in plant chloroplasts. Arabidopsis thaliana SAL1 (AtSAL1) senses changes in photosynthetic redox poise, hydrogen peroxide, and superoxide concentrations in chloroplasts via redox regulatory mechanisms. AtSAL1 phosphatase activity is suppressed by dimerization, intramolecular disulfide formation, and glutathionylation, allowing accumulation of its substrate, PAP, a chloroplast stress retrograde signal that regulates expression of plastid redox associated nuclear genes (PRANGs). This redox regulation of SAL1 for activation of chloroplast signaling is conserved in the plant kingdom, and the plant protein has evolved enhanced redox sensitivity compared with its yeast ortholog. Our results indicate that in addition to sulfur metabolism, SAL1 orthologs have evolved secondary functions in oxidative stress sensing in the plant kingdom.
See: http://www.pnas.org/content/113/31/E4567.abstract.html?etoc PNAS August 2, 2016; vol.113; no.31: E4567–E4576
Fig. 6. AtSAL1 is redox-regulated via intramolecular disulfide formation and dimerization in vivo, and it is sensitive to the chloroplast redox state. (A) Down-regulation of AtSAL1 activity and concomitant PAP accumulation correlates with formation of the Cys167–Cys190 intramolecular disulfide (black triangles) in endogenous AtSAL1 during drought stress. Means and SE are shown for n = 4 biological replicates for well-watered and n = 3 for drought. In contrast to Fig. 1A, leaf protein extracts were blocked with iodoacetamide, and then protein electrophoresis and Western blotting were performed under nonreducing conditions to visualize the Cys167–Cys190 disulfide. Loading control was Coomassie Blue staining. Similar results were obtained in two independent experiments. (B) The monomer–dimer equilibrium of AtSAL1 in vivo is shifted in favor of the dimer during oxidative stress, suggesting formation of the Cys119–Cys119 intermolecular disulfide to stabilize the dimer. Total leaf protein pooled from four biological replicates per treatment was resolved on Native-PAGE and immunoblotted, and the relative quantities of dimeric to monomeric AtSAL1 were estimated by image analysis on ImageJ. WW, well watered; MD, middrought; LD, late drought; HL, high light; MV, methyl viologen; H2O2, hydrogen peroxide. |
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