Abstract
Higher plants contain a small, 5-member family of Rieske non-heme oxygenases that comprise the inner plastid envelope protein TIC55, phaeophorbide a oxygenasee (PAO), chlorophyllide a oxygenase (CAO), choline monooxygenase, and a 52 kDa protein (PTC52) associated with the precursor NADPH:protochlorophyllide (Pchlide) oxidoreductase A (pPORA) A translocon (PTC). Some of these chloroplast proteins have documented roles in chlorophyll biosynthesis (CAO) and degradation (PAO and TIC55), whereas the function of PTC52 remains unresolved. Biochemical evidence provided here identifies PTC52 as Pchlide a oxygenase of the inner plastid envelope linking Pchlide b synthesis to pPORA import. Protochlorophyllide b is the preferred substrate of PORA and its lack no longer allows pPORA import. The Pchlide b-dependent import pathway of pPORA thus operates in etiolated seedlings and is switched off during greening. Using dexamethasone-induced RNA interference (RNAi) we tested if PTC52 is involved in controlling both, pPORA import and Pchlide homeostasis in planta. As shown here, RNAi plants deprived of PTC52 transcript and PTC52 protein were unable to import pPORA and died as a result of excess Pchlide a accumulation causing singlet oxygen formation during greening. In genetic studies, no homozygous ptc52 knock-out mutants could be obtained presumably as a result of embryo lethality, suggesting a role for PTC52 in the initial greening of plant embryos. Phylogenetic studies identified PTC52-like genes amongst unicellular photosynthetic bacteria and higher plants, suggesting that the biochemical function associated with PTC52 may have an ancient evolutionary origin. PTC52 also harbors conserved motifs with bacterial oxygenases such as the terminal oxygenase component of 3-ketosteroid 9-alpha-hydroxylase (KshA) from Rhodococcus rhodochrous. 3D-modeling of PTC52 structure permitted the prediction of amino acid residues that contribute to the substrate specificity of this enzyme. In vitro-mutagenesis was used to test the predicted PTC52 model and provide insights into the reaction mechanism of this Rieske non-heme oxygenase.
Highlights
The endosymbiotic theory explains the origin of mitochondria and plastids through the engulfment of bacteria into eukaryotic cells (Margulis, 1970)
PTC52 of barley was isolated by its co-purification with protochlorophyllide (Pchlide) oxidoreductase A (pPORA) in import intermediates trapped in junction complexes between the outer and inner plastid envelope membranes at 0.1 mM Mg-ATP and 0.1 mM Mg-GTP (Reinbothe et al, 2004a)
PTC52 Is Involved in Pchlide b Synthesis and pPORA Import
Summary
The endosymbiotic theory explains the origin of mitochondria and plastids through the engulfment of bacteria into (proto) eukaryotic cells (Margulis, 1970). 90% of the total gene transfer from the genome of the cyanobacterial endosymbiont to the host cell nucleus had taken place and after an envelope protein import machinery had evolved that allowed the gene products to be imported back into the semiautonomous photosynthetic organelle (Martin and Müller, 1998; Martin et al, 1998). It was for a long time believed that most of the proteins destined to the primordial chloroplast acquired cleavable NH2-terminal transit sequences for import (Keegstra et al, 1989). Evidence is emerging for the dual targeting of cytosolic proteins to mitochondria and chloroplasts (Peeters and Small, 2001), for the plastid import of transit peptide-less precursors (Miras et al, 2002, 2007; Nada and Soll, 2004; Rossig et al, 2013, 2014), and for the involvement of the secretory pathway in the import of certain precursors into chloroplasts (Villarejo et al, 2005; Baslam et al, 2016)
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