Chloroplast biogenesis: The use of mutants to study the etioplast–chloroplast transition

Abstract

In angiosperm plants, the etioplast-chloroplast transition is lightdependent. A key factor in this process is the protochlorophyllide oxidoreductase A (PORA), which catalyzes the light-induced reduction of protochlorophyllide to chlorophyllide. The import pathway of the precursor protein prePORA into chloroplasts was analyzed in vivo and in vitro by using homozygous loss-of-function mutants in genes coding for chlorophyllide a oxygenase (CAO) or for members of the outer-envelope solute-channel protein family of 16 kDa (OEP16), both of which have been implied to be key factors for the import of prePORA. Our in vivo analyses show that cao or oep16 mutants contain a normally structured prolamellar body that contains the protochlorophyllide holochrome. Furthermore, etioplasts from cao and oep16 mutants contain PORA protein as found by mass spectrometry. Our data demonstrate that both CAO and OEP16 are dispensable for chloroplast biogenesis and play no central role in the import of prePORA in vivo and in vitro as further indicated by protein import studies.chlorophyllide a oxygenase ͉ chloroplast outer-envelope solute channel ͉ protein import ͉ protochlorophyllide oxidoreductase

A search for factors influencing etioplast–chloroplast transition

“…Further, they postulate that the loss of OEP16.1 results in a lack of PORA in etioplasts that leads to the accumulation of free Pchlide a and in turn photo-oxidative damage of seedlings during dark to light transition. In contrast to the results of Reinbothe and coworkers, in our study the identical T-DNA insertion line for OEP16.1 greened and developed normally under a day-night regime and showed wild-type etioplast to chloroplast transition (39).…”

“…At-OEP16.2 is expressed exclusively in plastids of late embryo development, early cotyledons, and pollen grains, whereas low levels of At-OEP16.4 transcripts are ubiquitously present. At-OEP16.1 is the most prominent isoform in the outer envelope membrane of Arabidopsis leaf chloroplasts (16,(39)(40)(41) and shows the highest sequence identity with OEP16 from pea and barley. In a study on chloroplast biogenesis using a mutant approach with single-and double-knockout lines for all plastid-localized Arabidopsis OEP16 isoforms we could show that none of the OEP16 isoforms is involved in prePORA import into cotyledon chloroplasts and etioplasts in vitro and in vivo (39).…”

“…Like these transporters, OEP16 forms 4 ␣-helical transmembrane domains (38). In Arabidopsis 3 genes are coding for OEP16 isoforms, named OEP16.1, OEP16.2, and OEP16.4, respectively (16,39). At-OEP16.2 is expressed exclusively in plastids of late embryo development, early cotyledons, and pollen grains, whereas low levels of At-OEP16.4 transcripts are ubiquitously present.…”

Chloroplast iron-sulfur cluster protein maturation requires the essential cysteine desulfurase CpNifS

“…Leaf tissue for protein analysis was collected 10 days after induction. Antibodies for CpNifS (9), CpSufE (11), CpIscA (17), Fd and SiR (31), cytochrome f, light-harvesting complex of PSI, and PsaA/B (32), Fd-GOGAT and NiR (33), cytochrome b 6 and the Rieske subunit (34), PsaC and PsaD (35), protochlorophyllide reductase B and the small subunit of Rubisco (36), and the chloroplastic RRF (37) have been described. Specific antibodies for the D1 subunit of PSII and subunit A of ATP synthase were generous gifts from Alice Barkan (University of Oregon, Eugene) and Anna Sokolenko (Ludwig-Maximilians University, München, Germany), respectively.…”

A Pentapeptide Motif Related to a Pigment Binding Site in the Major Light-Harvesting Protein of Photosystem II, LHCII, Governs Substrate-Dependent Plastid Import of NADPH:Protochlorophyllide Oxidoreductase A

“…1B). The reason for these constructions lay in the observation that a fraction of the full-length pPORA synthesized in wheat germ extracts was targeted to Pchlide-free chloroplasts Dahlin et al, 2000;Philippar et al, 2007). A 14:3:3 protein recognition motif is present in the mature region of pPORA that governs Pchlide-independent default import (Schemenewitz et al, 2007).…”