Chloroplast biogenesis: the use of mutants to study the etioplast-chloroplast transition - PubMed (original) (raw)
Chloroplast biogenesis: the use of mutants to study the etioplast-chloroplast transition
Katrin Philippar et al. Proc Natl Acad Sci U S A. 2007.
Abstract
In angiosperm plants, the etioplast-chloroplast transition is light-dependent. 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.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Homozygous cao-1 mutants do not contain Chl b or Pchlide b. Eight-day-old seedlings of Col-0 wild-type (a and b) and cao-1 (c and d) were used for the extraction of pigments either without ALA (a and c) or after the addition of ALA (b and d) for 16 h in the dark. Arrowheads indicate the expected occurrence of Pchlide b. HPLC runs were monitored with a diode array detector. The relative absorbance at wavelengths of 430 nm (Chl a), 448 nm (Pchlide a/b), and 460 nm (Chl b) is depicted. Note that the extraction protocol used for the enrichment of Pchlide results in separation of most of Chl a and Chl b before HPLC analysis, resulting in different amounts of residual Chl (see Materials and Methods).
Fig. 2.
Import of prePORA and prePORB into chloroplasts is independent of Pchlide and can be competed for by preOE33. Chloroplasts were isolated from 8-day-old cotyledons of light-grown seedlings (light) or from plantlets harvested after a 16-h dark period, either without (dark) or with addition of ALA (dark + ALA). (a) Determination of Pchlide a from Col-0 wild-type (wt) and homozygous cao-1 plants grown under different conditions as outlined above. The Pchlide a content (n = 2) is given in nanomoles per gram of fresh weight (FW). (b) Import of prePORA and prePORB into chloroplasts from wild-type and cao-1 plantlets, either light-grown or treated with ALA (see above). Chloroplasts equivalent to 10 μg of Chl were incubated with the in vitro translated precursor proteins (Tl). After import, chloroplasts were either not treated (−) or treated (+) with the protease thermolysin. Precursor and mature proteins are indicated by asterisks and arrowheads, respectively. The letter A or B above each lane denotes the prePOR isoform used. (c) The import of prePORA and prePORB was tested in wild-type chloroplasts using urea-denatured precursor proteins (urea) or sucrose as an osmoticum during chloroplast isolation (Suc). All other conditions were as described for b. (d) The import of preSSU, prePORA, and prePORB can be competed for by preOE33 but not by mOE33. OE33 proteins were heterologously expressed as soluble proteins and added at a final concentration of 5 μM to the import reaction before precursor proteins. Import rates were determined by measuring the signal density of the respective mature radiolabeled protein. Signals of imports with added competitor were normalized to the signals in control imports (without OE33), which were set to 1.0 (arbitrary units). A mean import rate ± SD of three (prePORA and prePORB) and five (preSSU) independent reactions is shown.
Fig. 3.
Differential and developmental occurrence of OEP16 isoforms in Arabidopsis. (a) Western blot analysis of the distribution of OEP16 isoforms between chloroplasts (C) and mitochondria (M). Except for the detection of OEP16.2 in chloroplasts of 8-day-old cotyledons, organelles from leaves of 3-week-old plants were used. Antisera against the marker proteins Toc159, OEP37, and VDAC (outer membrane of mitochondria) were used as controls. Numbers indicate the molecular mass of proteins in kDa. (b) The presence of OEP16.1 and OEP16.2 was tested by immunoblot analysis of dry seeds and during germination in 0-, 2-, 4-, 6-, and 8-day-old seedlings. (c) Presence of POR and OEP16.1 proteins in 8-day-old green (lane 1) and etiolated (et; lane 2) cotyledons and during the exposure of etiolated cotyledons to light for the times indicated (lanes 3–6). POR polypeptides are highly identical; therefore, the antibody used recognizes all POR isoforms in Arabidopsis. Please note that OEP16.2 is absent in light-grown and etiolated 8-day-old seedlings (see b, rightmost lane) and therefore was not detectable during the deetiolation of cotyledons.
Fig. 4.
Etioplast-to-chloroplast transition in Arabidopsis cotyledons. Plastids are shown from etiolated seedlings (Left), after exposure to light for 16 h (Center), or grown under a normal day-night cycle (Right). (a) Col-0 wild-type control. (b) Homozygous oep16.1/16.4 double knockout mutant. (Scale bars: 2.5 μm.)
Fig. 5.
Import of prePORA and prePORB is not impaired in chloroplasts from oep16 knockout mutants. (a) Typical import reactions are shown to compare the import yield of prePORA and prePORB into chloroplasts from Col-0 wild-type (lanes 3–6) or oep16 mutants (lanes 7–10) as indicated. Chloroplasts equivalent to 10 μg of Chl were incubated with the _in-vitro_-translated precursor proteins (Tl). After import, chloroplasts were either not treated (−) or treated (+) with the protease thermolysin. Precursor and mature proteins are indicated by asterisks and arrowheads, respectively. (b) Import yield of prePORA and prePORB into chloroplasts isolated from light-grown or ALA-treated (dark + ALA) oep16 knockout mutants. All other conditions were as outlined in a and in Fig. 2 b and c.
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