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The enzyme protochlorophyllide oxidoreductase (POR) catalyses a light-dependent step in chlorophyll biosynthesis that is essential to photosynthesis and, ultimately, all life on Earth 1 – 3 . POR, which is one of three known light-dependent enzymes 4 , 5 , catalyses reduction of the photosensitizer and substrate protochlorophyllide to form the pigment chlorophyllide. Despite its biological importance, the structural basis for POR photocatalysis has remained unknown. Here we report crystal structures of cyanobacterial PORs from Thermosynechococcus elongatus and Synechocystis sp. in their free forms, and in complex with the nicotinamide coenzyme. Our structural models and simulations of the ternary protochlorophyllide–NADPH–POR complex identify multiple interactions in the POR active site that are important for protochlorophyllide binding, photosensitization and photochemical conversion to chlorophyllide. We demonstrate the importance of active-site architecture and protochlorophyllide structure in driving POR photochemistry in experiments using POR variants and protochlorophyllide analogues. These studies reveal how the POR active site facilitates light-driven reduction of protochlorophyllide by localized hydride transfer from NADPH and long-range proton transfer along structurally defined proton-transfer pathways. Crystal structures of cyanobacterial protochlorophyllide oxidoreductases reveal the basis of the photocatalytic activities of this enzyme, through the role of its active site in enabling the light-driven reduction of protochlorophyllide.

Rice (Oryza sativa L.) frequently suffers in late spring from severe damage due to cold spells, which causes the block of chlorophyll biosynthesis during early rice seedling greening. However, the inhibitory mechanism by which this occurs is still unclear. To explore the responsive mechanism of rice seedlings to low temperatures during greening, the effects of chilling stress on chlorophyll biosynthesis and plastid development were studied in rice seedlings. Chlorophyll biosynthesis was obviously inhibited and chlorophyll accumulation declined under low temperatures during greening. The decrease in chlorophyll synthesis was due to the inhibited synthesis of δ-aminolevulinic acid (ALA) and the suppression of conversion from protochlorophyllide (Pchlide) into chlorophylls (Chls). Meanwhile, the activities of glutamate-1-semialdehyde transaminase (GSA-AT), Mg-chelatase, and protochlorophyllide oxidoreductase (POR) were downregulated under low temperatures. Further investigations showed that chloroplasts at 18 °C had loose granum lamellae, while the thylakoid and lamellar structures of grana could hardly develop at 12 °C after 48 h of greening. Additionally, photosystem II (PSII) and photosystem I (PSI) proteins obviously declined in the stressed seedlings, to the point that the PSII and PSI proteins could hardly be detected after 48 h of greening at 12 °C. Furthermore, the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA) and cell death were all induced by low temperature. Chilling stress had no effect on the development of epidermis cells, but the stomata were smaller under chilling stress than those at 28 °C. Taken together, our study promotes more comprehensive understanding in that chilling could inhibit chlorophyll biosynthesis and cause oxidative damages during greening.

Angiosperms germinated in the dark develop etioplasts, the chloroplast precursors, in cotyledon cells. Etioplasts contain lattice membrane structures called prolamellar bodies (PLBs) and lamellar prothylakoids as internal membrane systems. PLBs accumulate the chlorophyll intermediate protochlorophyllide (Pchlide) in a complex with NADPH and light-dependent NADPH:Pchlide oxidoreductase (LPOR). Two galactolipids, monogalactosyldiacylglycerol and digalactosyldiacylglycerol (DGDG), are major constituents of etioplast membranes. We previously reported that monogalactosyldiacylglycerol facilitates the synthesis of Pchlide and the formation of the Pchlide-LPOR-NADPH complex in etioplasts, but the importance of DGDG in etioplasts is still unknown. To determine the role of DGDG in etioplast development and functions, we characterized a knockout mutant (dgd1) of Arabidopsis (Arabidopsis thaliana) DGD1, which encodes the major isoform of DGDG synthase, in the etioplast development stage. In etiolated dgd1 seedlings, DGDG content decreased to 20% of the wild-type level, the lattice structure of PLBs was disordered, and the development of prothylakoids was impaired. In addition, membrane-associated processes of Pchlide biosynthesis, formation of the Pchlide-LPOR-NADPH complex, and dissociation of the complex after the photoconversion of Pchlide to chlorophyllide were impaired in dgd1, although the photoconversion reaction by LPOR was not affected by the DGDG deficiency. Total carotenoid content also decreased in etiolated dgd1 seedlings, but the carotenoid composition was unchanged. Our data demonstrate a deep involvement of DGDG in the formation of the internal membrane structures in etioplasts as well as in membrane-associated processes of pigment biosynthesis and pigment-protein complex organization.

Malus halliana is an iron (Fe)-efficient apple rootstock growing in calcareous soil that shows obvious 'greenness' traits during Fe deficiency. Recent studies have shown that exogenous sugars can be involved in abiotic stress. To identify the key regulatory steps of chlorophyll (Chl) biosynthesis in M. halliana under Fe deficiency and to verify whether exogenous sucrose (Suc) is involved in Fe deficiency stress, we determined the contents of the Chl precursor and the expression of several Chl biosynthetic genes in M. halliana. The results showed that Fe deficiency caused a significant increase in the contents of protoporphyrin IX (Proto IX), Mg-protoporphyrin IX (Mg-Proto IX) and protochlorophyllide (Pchlide) in M. halliana compared to the Fe-sensitive rootstock Malus hupehensis. Quantitative real-time PCR (RT-qPCR) also showed that the expression of protoporphyrinogen oxidase (PPOX), which synthesizes Proto IX, was upregulated in M. halliana and downregulated in M. hupehensis under Fe deficiency. Exogenous Suc application prominently enhanced the contents of porphobilinogen (PBG) and the subsequent precursor, whereas it decreased the level of δ-aminolaevulinic acid (ALA), suggesting that the transformation from ALA to PBG was catalyzed in M. halliana. Additionally, the transcript level of δ-aminolevulinate acid dehydratase (ALAD) was noticeably upregulated after exogenous Suc treatment. This result, combined with the precursor contents, indicated that Suc accelerated the steps of Chl biosynthesis by modulating the ALAD gene. Therefore, we conclude that PPOX is the key regulatory gene of M. halliana in response to Fe deficiency. Exogenous Suc enhances M. halliana tolerance to Fe deficiency stress by regulating Chl biosynthesis.

In plants, light represents an important environmental signal that triggers the production of photosynthetically active chloroplasts. This developmental switch is critical for plant survival because chlorophyll precursors that accumulate in darkness can be extremely destructive when illuminated. Thus, plants have evolved mechanisms to adaptively control plastid development during the transition into light. Here, we report that the gibberellin (GA)-regulated DELLA proteins play a crucial role in the formation of functional chloroplasts during deetiolation. We show that Arabidopsis thaliana DELLAs accumulating in etiolated cotyledons derepress chlorophyll and carotenoid biosynthetic pathways in the dark by repressing the transcriptional activity of the phytochrome-interacting factor proteins. Accordingly, dark-grown GA-deficient ga1-3 mutants (that accumulate DELLAs) display a similar gene expression pattern to wild-type seedlings grown in the light. Consistent with this, ga1-3 seedlings accumulate higher amounts of protochlorophyllide (a phototoxic chlorophyll precursor) in darkness but, surprisingly, are substantially more resistant to photooxidative damage following transfer into light. This is due to the DELLA-dependent upregulation of the photoprotective enzyme protochlorophyllide oxidoreductase (POR) in the dark. Our results emphasize the role of DELLAs in regulating the levels of POR, protochlorophyllide, and carotenoids in the dark and in protecting etiolated seedlings against photooxidative damage during initial light exposure.

Enzymes that catalyze long-range electron transfer (ET) reactions often function as higher order complexes that possess two structurally symmetrical halves. The functional advantages for such an architecture remain a mystery. Using cryoelectron microscopy we capture snapshots of the nitrogenase-like dark-operative protochlorophyllide oxidoreductase (DPOR) during substrate binding and turnover. DPOR catalyzes reduction of the C17 = C18 double bond in protochlorophyllide during the dark chlorophyll biosynthetic pathway. DPOR is composed of electron donor (L-protein) and acceptor (NB-protein) component proteins that transiently form a complex in the presence of ATP to facilitate ET. NB-protein is an α 2 β 2 heterotetramer with two structurally identical halves. However, our structures reveal that NB-protein becomes functionally asymmetric upon substrate binding. Asymmetry results in allosteric inhibition of L-protein engagement and ET in one half. Residues that form a conduit for ET are aligned in one half while misaligned in the other. An ATP hydrolysis-coupled conformational switch is triggered once ET is accomplished in one half. These structural changes are then relayed to the other half through a di-nuclear copper center at the tetrameric interface of the NB-protein and leads to activation of ET and substrate reduction. These findings provide a mechanistic blueprint for regulation of long-range electron transfer reactions. CryoEM snapshots of the nitrogenase-like DPOR protein complex captured during turnover reveal that asymmetric conformational changes, substrate recognition, and an interplay between copper and iron sulfur metalloclusters is required for electron transfer.

5-Aminolevulinic acid (ALA) is the first committed substrate of tetrapyrrole biosynthesis and is formed from glutamyl-tRNA by two enzymatic steps. Glutamyl-tRNA reductase (GluTR) as the first enzyme of ALA synthesis is encoded by HEMA genes and tightly regulated at the transcriptional and posttranslational levels. Here, we show that the caseinolytic protease (Clp) substrate adaptor ClpS1 and the ClpC1 chaperone as well as the GluTR-binding protein (GBP) interact with the N terminus of GluTR. Loss-of function mutants of ClpR2 and ClpC1 proteins show increased GluTR stability, whereas absence of GBP results in decreased GluTR stability. Thus, the Clp protease system and GBP contribute to GluTR accumulation levels, and thereby the rate-limiting ALA synthesis. These findings are supported with Arabidopsis (Arabidopsis thaliana) hema1 mutants expressing a truncated GluTR lacking the 29 N-terminal amino acid residues of the mature protein. Accumulation of this truncated GluTR is higher in dark periods, resulting in increased protochlorophyllide content. It is proposed that the proteolytic activity of Clp protease counteracts GBP binding to assure the appropriate content of GluTR and the adequate ALA synthesis for chlorophyll and heme in higher plants.

3,8-Divinyl (proto)chlorophyll(ide) a 8-vinyl reductase (DVR) catalyzes the reduction of 8-vinyl group on the tetrapyrrole to an ethyl group, which is indispensable for monovinyl chlorophyll (Chl) synthesis. So far, three 8-vinyl reductase genes (DVR, bciA, and slr1923) have been characterized from Arabidopsis (Arabidopsis thaliana), Chlorobium tepidum, and Synechocystis sp. PCC6803. However, no 8-vinyl reductase gene has yet been identified in monocotyledonous plants. In this study, we isolated a spontaneous mutant, 824ys, in rice (Oryza sativa). The mutant exhibited a yellow-green leaf phenotype, reduced Chl level, arrested chloroplast development, and retarded growth rate. The phenotype of the 824ys mutant was caused by a recessive mutation in a nuclear gene on the short arm of rice chromosome 3. Map-based cloning of this mutant resulted in the identification of a gene (Os03g22780) showing sequence similarity with the Arabidopsis DVR gene (AT5G18660). In the 824ys mutant, nine nucleotides were deleted at residues 952 to 960 in the open reading frame, resulting in a deletion of three amino acid residues in the encoded product. High-performance liquid chromatography analysis of Chls indicated the mutant accumulates only divinyl Chl a and b. A recombinant protein encoded by Os03g22780 was expressed in Escherichia coli and found to catalyze the conversion of divinyl chlorophyll(ide) a to monovinyl chlorophyll(ide) a. Therefore, it has been confirmed that Os03g22780, renamed as OsDVR, encodes a functional DVR in rice. Based upon these results, we succeeded to identify an 8-vinyl reductase gene in monocotyledonous plants and, more importantly, confirmed the DVR activity to convert divinyl Chl a to monovinyl Chl a.

The LIL3 protein of Arabidopsis (Arabidopsis thaliana) belongs to the light-harvesting complex (LHC) protein family, which also includes the light-harvesting chlorophyll-binding proteins of photosystems I and II, the early-light-inducible proteins, PsbS involved in nonphotochemical quenching, and the one-helix proteins and their cyanobacterial homologs designated high-light-inducible proteins. Each member of this family is characterized by one or two LHC transmembrane domains (referred to as the LHC motif) to which potential functions such as chlorophyll binding, protein interaction, and integration of interacting partners into the plastid membranes have been attributed. Initially, LIL3 was shown to interact with geranylgeranyl reductase (CHLP), an enzyme of terpene biosynthesis that supplies the hydrocarbon chain for chlorophyll and tocopherol. Here, we show another function of LIL3 for the stability of protochlorophyllide oxidoreductase (POR). Multiple protein-protein interaction analyses suggest the direct physical interaction of LIL3 with POR but not with chlorophyll synthase. Consistently, LIL3-deficient plants exhibit substantial loss of POR as well as CHLP, which is not due to defective transcription of the POR and CHLP genes but to the posttranslational modification of their protein products. Interestingly, in vitro biochemical analyses provide novel evidence that LIL3 shows high binding affinity to protochlorophyllide, the substrate of POR. Taken together, this study suggests a critical role for LIL3 in the organization of later steps in chlorophyll biosynthesis. We suggest that LIL3 associates with POR and CHLP and thus contributes to the supply of the two metabolites, chlorophyllide and phytyl pyrophosphate, required for the final step in chlorophyll a synthesis.

In buried seedlings, chloroplasts are arrested at the etioplast stage, but they rapidly mature upon emergence of the seedling. Etioplast-chloroplast differentiation is halted through the integration of soil-induced signals, including pressure and the absence of light, although the details on how this information converges to regulate cellular decisions remain unclear. Here, we identify an interdependent transcription module that integrates the mechanical pressure and darkness signals to control chloroplast development in Arabidopsis thaliana. Mutations of ETHYLENE-INSENSITIVE3 (EIN3), the primary transcription factor in the ethylene signaling pathway that is activated in response to mechanical pressure, cause early development of etioplasts in the dark and severe photobleaching upon light exposure. Genetic studies demonstrate that repression of etioplast differentiation by EIN3 requires PHYTOCHROME INTERACTING FACTOR3 (PIF3), a darkness-stabilized bHLH transcription factor. EIN3 and PIF3 directly interact and form an interdependent module to repress the expression of most LIGHT HARVESTING COMPLEX (LHC) genes; overexpressing even one LHC could cause premature development of etioplasts. The EIN3-PIF3 transcription module synergistically halts chloroplast development by interdependently co-occupying the promoters of LHC genes. Thus, our results define a transcriptional regulatory module and provide mechanistic insight on the concerted regulation of chloroplast development by multiple soil-induced signals.