Cell growth defect factor 1 is crucial for the plastid import of NADPH:protochlorophyllide oxidoreductase A in Arabidopsis thaliana

Tetrapyrroles such as chlorophyll, heme, and bacteriochlorophyll play fundamental roles in the energy absorption and transduction of all photosynthetic organisms. They are synthesized via a complex pathway taking place in chloroplasts. Chlorophyll biosynthesis in angiosperms involves 16 steps of which only one is light-requiring and driven by the NADPH:protochlorophyllide oxidoreductase (POR). Three POR isoforms have been identified in Arabidopsis thaliana—designated PORA, PORB, and PORC—that are differentially expressed in etiolated, light-exposed, and light-adapted plants. All three isoforms are encoded by nuclear genes, are synthesized as larger precursors in the cytosol (pPORs), and are imported posttranslationally into the plastid compartment. Import of the precursor to the dark-specific isoform PORA (pPORA) is protochlorophyllide (Pchlide)-dependent and due to the operation of a unique translocon complex dubbed PTC (Pchlide-dependent translocon complex) in the plastid envelope. Here, we identified a ∼30-kDa protein that participates in pPORA import. The ∼30-kDa protein is identical to the previously identified CELL GROWTH DEFECT FACTOR 1 (CDF1) in Arabidopsis that is conserved in higher plants and Synechocystis. CDF1 operates in pPORA import and stabilization and hereby acts as a chaperone for PORA protein translocation. CDF1 permits tight interactions between Pchlide synthesized in the plastid envelope and the importing PORA polypeptide chain such that no photoexcitative damage occurs through the generation of singlet oxygen operating as a cell death inducer. Together, our results identify an ancient mechanism dating back to the endosymbiotic origin of chloroplasts as a key element of Pchlide-dependent pPORA import.Higher plants make use of two closely related forms of chlorophyll (Chl) for light harvesting and energy transduction, Chl a and Chl b. Both are synthesized from 5-aminolevulinic acid (5-ALA) (see refs. 1, 2 for review). Chl biosynthesis is controlled at several different levels in angiosperms, including (i) feedback inhibition by heme of the early steps leading to 5-ALA (3, 4), (ii) repression of Chl precursor accumulation at the level of protochlorophyllide (Pchlide) by the FLUORESCENT protein (5), and (iii) light activation of Pchlide to chlorophyllide (Chlide) conversion by the NADPH:Pchlide oxidoreductase (POR) (6).In Arabidopsis, three differentially expressed POR isoenzymes exist, named PORA, PORB, and PORC (summarized in ref. 6). Mutagenesis and homology modeling studies have provided important insights into the catalytic mechanism of POR (7–11). All three POR isoforms are nuclear gene products that must be imported posttranslationally into the plastids (12, 13). Components have been identified in the plastid envelope mediating this import step (14–16). Interestingly, it could be demonstrated that PORA and PORB use different pathways for import (17). Whereas transport of the pPORA is Pchlide-dependent and occurs through a unique import site, uptake of the pPORB occurs through the general import site (14–18). The pPORA and pPORB contain structurally distinct NH₂-terminal transit peptides that direct the precursors to the different protein import machineries in the chloroplast envelope (14–18).The question of how pPORA translocation is mechanistically coupled to Pchlide synthesis in the plastid envelope has not been answered yet. Here, we identified a protein interacting with pPORA during its Pchlide-dependent import and show that it is related to a previously identified cell death factor, named CELL GROWTH DEFECT FACTOR 1 (CDF1) (19). Suppression of Arabidopsis thaliana CDF1 (AtCDF1) expression led to a complete block of pPORA import into etioplasts, overaccumulation of non–protein-bound Pchlide, and light-dependent, porphyrin-sensitized cell death involving singlet oxygen. Along with biochemical data showing tight protein:protein interactions between CDF1 and PORA, we propose CDF1 to act as a POR-specific chaperone during plastid biogenesis.