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

Abstract

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.

Common functions of the chloroplast and mitochondrial co-chaperones cpDnaJL (CDF1) and mtDnaJ (PAM16) in protein import and ROS scavenging in Arabidopsis thaliana

“…Lack of cpDnaJL, as found after RNA interference-induced gene silencing ( RNAi ), impaired the establishment of these complexes (Fig. 3) and led to overaccumulation of free Pchlide acting as photosensitizer for singlet oxygen formation during greening 10 . Measurements with the dansyl-based singlet oxygen quencher DanePy confirmed mass generation of ROS in cpDnaJL-depleted RNAi seedlings 10 .…”

“…Measurements with the dansyl-based singlet oxygen quencher DanePy confirmed mass generation of ROS in cpDnaJL-depleted RNAi seedlings 10 . Accumulation of singlet oxygen and other ROS explains why young-born sprouts rapidly die during greening 10 . Interestingly, similar, essential functions of mtDnaJ in mitochondrial biogenesis and function were deduced from knock-out mutant studies using different PAM16 alleles 12,13 .…”

“…cpDnaJL does not seem to participate in overall import of presequence-containing or presequence-less cytosolic precursors; instead it accomplishes a specific role. cpDnaJL is part of a unique protein import site through which NADPH:protochlorophyllide oxidoreductase (POR) A is specifically imported 10 (Fig. 2B).…”

“…Uptake of the pPORA into the plastids is unique in requiring protochlorophyllide (Pchlide) as import trigger 14-17 . Time courses over import revealed that the pPORA sequentially binds the tetrapyrrole pigment Pchlide 10 and thereby lowers its interactions with molecular oxygen provoking the generation of singlet oxygen as cytotoxin and cell death inducer 18,19 . The interaction between the translocating pPORA polypeptide chain and Pchlide is enabled by the binding of cpDnaJL that operates independently of cpHSP70 and keeps the pPORA in a state ready to bind Pchlide 9,10 Once pPORA's transit sequence is removed by the stromal processing peptidase, the mature pPORA loaded with Pchlide interacts with PORB and assembles into larger light-harvesting complexes in the prolamellar body of etiolated plants 20,21 .…”

“…3) and led to overaccumulation of free Pchlide acting as photosensitizer for singlet oxygen formation during greening 10 . Measurements with the dansyl-based singlet oxygen quencher DanePy confirmed mass generation of ROS in cpDnaJL-depleted RNAi seedlings 10 . Accumulation of singlet oxygen and other ROS explains why young-born sprouts rapidly die during greening 10 .…”