Abstract
NADPH is a key reductant carrier that maintains internal redox and antioxidant status, and that links biosynthetic, catabolic and signalling pathways. Plants have a mitochondrial external NADPH oxidation pathway, which depends on Ca2+ and pH in vitro, but concentrations of Ca2+ needed are not known. We have determined the K0.5(Ca2+) of the external NADPH dehydrogenase from Solanum tuberosum mitochondria and membranes of E. coli expressing Arabidopsis thaliana NDB1 over the physiological pH range using O2 and decylubiquinone as electron acceptors. The K0.5(Ca2+) of NADPH oxidation was generally higher than for NADH oxidation, and unlike the latter, it depended on pH. At pH 7.5, K0.5(Ca2+) for NADPH oxidation was high (≈100 μM), yet 20-fold lower K0.5(Ca2+) values were determined at pH 6.8. Lower K0.5(Ca2+) values were observed with decylubiquinone than with O2 as terminal electron acceptor. NADPH oxidation responded to changes in Ca2+ concentrations more rapidly than NADH oxidation did. Thus, cytosolic acidification is an important activator of external NADPH oxidation, by decreasing the Ca2+-requirements for NDB1. The results are discussed in relation to the present knowledge on how whole cell NADPH redox homeostasis is affected in plants modified for the NDB1 gene.
Highlights
Ca2+ has a central function in signalling in plant cells, being regulatory in several intrinsic and extrinsic conditions including abiotic and biotic stress responses, stomatal closure, tip growth and photoreceptor signalling [1]
The inner membrane integrity of isolated mitochondria from potato tubers was tested as the Alm permeabilisation-dependence of EGTA-insensitive NADH oxidation, the activity of which depends on the access of NADH to membrane-bound DHs oriented only towards the matrix
We observed a high maximum activity of 480 ± 85 nmol min-1 mg protein-1 and an Alm latency of 94 ± 1.5%. This indicates that the inner membranes of the mitochondria were practically impermeable to reduced pyridine nucleotides, and that a measured NADPH oxidation in the absence of Alm would only be due to the activity of the external NADPH DH
Summary
Ca2+ has a central function in signalling in plant cells, being regulatory in several intrinsic and extrinsic conditions including abiotic and biotic stress responses, stomatal closure, tip growth and photoreceptor signalling [1]. The cytosolic Ca2+ concentration will in an unstimulated or “resting” cell reside at a low level of around 100 nM. In response to stimuli, oscillatory increases in the cytosolic Ca2+ concentration occur. The amplitude and period depend on stimuli and cell type, each combination having its specific Ca2+ signature [2]. Cytosolic Ca2+ concentrations as high as 5 μM or higher have been recorded in response to external stress [3]. In addition to major effects on gene expression [4], Ca2+ interacts directly with the PLOS ONE | DOI:10.1371/journal.pone.0139224. In addition to major effects on gene expression [4], Ca2+ interacts directly with the PLOS ONE | DOI:10.1371/journal.pone.0139224 September 28, 2015
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