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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.

The nonhomologous enzymes, the light-independent protochlorophyllide reductase (DPOR) and the light-dependent protochlorophyllide oxidoreductase (LPOR), catalyze the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) in the penultimate step of biosynthesis of chlorophyll (Chl) required for photosynthetic light absorption and energy conversion. The two enzymes differ with respect to the requirement of light for catalysis and oxygen sensitivity. DPOR and LPOR initially evolved in the ancestral prokaryotic genome perhaps at different times. DPOR originated in the anoxygenic environment of the Earth from nitrogenase-like enzyme of methanogenic archaea. Due to the transition from anoxygenic to oxygenic photosynthesis in the prokaryote, the DPOR was mostly inactivated in the daytime by photosynthetic O2 leading to the evolution of oxygen-insensitive LPOR that could function in the light. The primary endosymbiotic event transferred the DPOR and LPOR genes to the eukaryotic phototroph; the DPOR remained in the genome of the ancestor that turned into the plastid, whereas LPOR was transferred to the host nuclear genome. From an evolutionary point of view, several compelling theories that explain the disappearance of DPOR from several species cutting across different phyla are as follows: (i) pressure of the oxygenic environment; (ii) change in the light conditions and temperature; and (iii) lineage-specific gene losses, RNA editing, and nonsynonymous substitution. Certain primary amino acid sequence and the physiochemical properties of the ChlL subunit of DPOR have similarity with that of LPOR suggesting a convergence of these two enzymes in certain evolutionary event. The newly obtained sequence data from different phototrophs will further enhance the width of the phylogenetic information on DPOR.

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.

The tocopherol biosynthetic pathway, encoded by VTE genes 1 through 6, is highly conserved in plants but most large effect quantitative trait loci for seed total tocopherols (totalT) lack VTE genes, indicating other activities are involved. A genome-wide association study of Arabidopsis seed tocopherols showed five of seven significant intervals lacked VTE genes, including the most significant, which mapped to an uncharacterized, seed-specific, envelope-localized, alpha/beta hydrolase with esterase activity, designated AtVTE7. Atvte7 null mutants decreased seed totalT 55% while a leaky allele of the maize ortholog, ZmVTE7, decreased kernel and leaf totalT 38% and 49%, respectively. Overexpressing AtVTE7 or ZmVTE7 partially or fully complemented the Atvte7 seed phenotype and increased leaf totalT by 3.6- and 6.9-fold, respectively. VTE7 has the characteristics of an esterase postulated to provide phytol from chlorophyll degradation for tocopherol synthesis, but bulk chlorophyll levels were unaffected in vte7 mutants and overexpressing lines. Instead, levels of specific chlorophyll biosynthetic intermediates containing partially reduced side chains were impacted and strongly correlated with totalT. These intermediates are generated by a membrane-associated biosynthetic complex containing protochlorophyllide reductase, chlorophyll synthase, geranylgeranyl reductase (GGR) and light harvesting-like 3 protein, all of which are required for both chlorophyll and tocopherol biosynthesis. We propose a model where VTE7 releases prenyl alcohols from chlorophyll biosynthetic intermediates, which are then converted to the corresponding diphosphates for tocopherol biosynthesis.

Germinated plant seeds buried in soil undergo skotomorphogenic development before emergence to reach the light environment. Young seedlings transitioning from dark to light undergo photomorphogenic development. During photomorphogenesis, light alters the transcriptome and enhances the translation of thousands of mRNAs during the dark-to-light transition in Arabidopsis young seedlings. About 1,500 of these mRNAs have comparable abundance before and after light treatment, which implies wide-spread translational repression in dark-grown seedlings. Processing bodies (p-bodies), the cytoplasmic granules found in diverse organisms, can balance the storage, degradation, and translation of mRNAs. However, the function of p-bodies in translation control remains largely unknown in plants. Here we found that an Arabidopsis mutant defective in p-body formation (Decapping 5; dcp5-1) showed reduced fitness under both dark and light conditions. Comparative transcriptome and translatome analyses of wild-type and dcp5-1 seedlings revealed that p-bodies can attenuate the premature translation of specific mRNAs in the dark, including those encoding enzymes for protochlorophyllide synthesis and PIN-LIKES3 for auxin-dependent apical hook opening. When the seedlings protrude from soil, light perception by photoreceptors triggers a reduced accumulation of p-bodies to release the translationally stalled mRNAs for active translation of mRNAs encoding proteins needed for photomorphogenesis. Our data support a key role for p-bodies in translation repression, an essential mechanism for proper skotomorphogenesis and timely photomorphogenesis in seedlings.

Manganese deficiency is a serious plant nutritional disorder, resulting in the loss of crop productivity in many parts of the world. Despite the progress made in the study of angiosperms, the demand for Mn in gymnosperms and the physiological responses to Mn deficiency remain unexplored. We studied the influence of Mn deficiency for 24 weeks on Pinus sylvestris L. seedling growth, ion homeostasis, pigment contents, lipid peroxidation, chlorophyll fluorescence indices and the transcript levels of photosynthetic genes and genes involved in chlorophyll biosynthesis. It was shown that Mn-deficient plants demonstrated suppressed growth when the Mn content in the needles decreased below 0.34 µmol/g DW. The contents of photosynthetic pigments decreased when the Mn content in the needles reached 0.10 µmol/g DW. Mn deficiency per se did not lead to a decrease in the nutrient content in the organs of seedlings. Photoinhibition of PSII was observed in Mn-deficient plants, although this was not accompanied by the development of oxidative stress. Mn-deficient plants had an increased transcript abundance of genes (psbO, psbP, psbQ, psbA and psbC), encoding proteins directly associated with the Mn cluster also as other proteins involved in photosynthesis, whose activities do not depend on Mn directly. Furthermore, the transcript levels of the genes encoding the large subunit of Rubisco, light-dependent NADPH-protochlorophyllide oxidoreductase and subunits of light-independent protochlorophyllide reductase were also increased in Mn-deficient 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 application of pesticides may adversely impact a variety of non-target organisms. The use of side-effect-free herbal remedies to protect against the toxicity of harmful pesticides such as etoxazole has gained attention in recent times. The current study aimed to reveal the potential mitigating efficacy of Achillea millefolium L. extract against etoxazole toxicity in Allium cepa L. A. cepa bulbs in the control group were applied with tap water, while bulbs in the treatment groups were applied with etoxazole at dose of 0.5 m/L and two different doses of A. millefolium extract (200 mg/L and 400 mg/L). The impact of the treatments on certain parameters was evaluated. The molecular docking analysis was employed to investigate the potential interactions of etoxazole with DNA species, DNA topoisomerases, tubulin proteins, glutamate-1-semialdehyde aminotransferase, and protochlorophyllide reductase. The phenolic profile of A. millefolium was assessed. Etoxazole exposure reduced rooting percentage, root length, weight gain, mitotic index, and levels of chlorophyll a and chlorophyll b. Conversely, etoxazole treatment led to an increase in chromosomal aberrations and micronuclei occurrence. The most frequently observed chromosomal aberrations induced by etoxazole, which serve as bioindicators of genotoxicity, were fragment, vagrant chromosome, sticky chromosome, unequal chromatin distribution, bridge, reverse polarization, and vacuolated nucleus. The levels of malondialdehyde and antioxidant enzyme (superoxide dismutase and catalase) activities were also elevated. Epidermis cell damage, flattened cell nucleus, thickened cortex cell wall, and thickened conduction tissue were the meristematic cell disorders triggered by etoxazole. Molecular docking studies showed that etoxazole can interact directly with DNA, tubulins, and the enzymes mentioned above. A. millefolium extract was found to contain a substantial quantity of phenolic compounds. A. millefolium extract, when co-administered with etoxazole, attenuated all toxic effects of etoxazole dose-dependently. In conclusion, A. millefolium may potentially serve as a reliable pharmacological shield against the toxicity of pesticides in non-target organisms.