Protochlorophyllide Oxidoreductase Research Articles

Curvature thylakoid 1 proteins modulate prolamellar body morphology and promote organized thylakoid biogenesis in Arabidopsis thaliana

The term "de-etiolation" refers to the light-dependent differentiation of etioplasts to chloroplasts in angiosperms. The underlying process involves reorganization of prolamellar bodies (PLBs) and prothylakoids into thylakoids, with concurrent changes in protein, lipid, and pigment composition, which together lead to the assembly of active photosynthetic complexes. Despite the highly conserved structure of PLBs among land plants, the processes that mediate PLB maintenance and their disassembly during de-etiolation are poorly understood. Among chloroplast thylakoid membrane-localized proteins, to date, only Curvature thylakoid 1 (CURT1) proteins were shown to exhibit intrinsic membrane-bending capacity. Here, we show that CURT1 proteins, which play a critical role in grana margin architecture and thylakoid plasticity, also participate in de-etiolation and modulate PLB geometry and density. Lack of CURT1 proteins severely perturbs PLB organization and vesicle fusion, leading to reduced accumulation of the light-dependent enzyme protochlorophyllide oxidoreductase (LPOR) and a delay in the onset of photosynthesis. In contrast, overexpression of CURT1A induces excessive bending of PLB membranes, which upon illumination show retarded disassembly and concomitant overaccumulation of LPOR, though without affecting greening or the establishment of photosynthesis. We conclude that CURT1 proteins contribute to the maintenance of the paracrystalline PLB morphology and are necessary for efficient and organized thylakoid membrane maturation during de-etiolation.

Unravelling the Role of Piriformospora indica in Combating Water Deficiency by Modulating Physiological Performance and Chlorophyll Metabolism-Related Genes in Cucumis sativus

Water stress is the most critical aspect restricting the development of agriculture in regions with scarce water resources, which requires enhancing irrigation water-saving strategies. The current work discusses the potential application of the plant-strengthening root endophyte Piriformospora indica against moderate (25% less irrigation water) and severe (50% less irrigation water) water stress in comparison to the optimum irrigation conditions of greenhouse cucumbers. P. indica improved growth, nutrient content, and photosynthesis apparatus under normal or water-stress conditions. On the other hand, moderate and severe water stress reduced yield up to 47% and 83%, respectively, in non-colonized cucumber plants, while up to 28 and 78%, respectively, in P. indica-colonized plants. In terms of water-use efficiency (WUE), P. indica improved the WUE of colonized cucumber plants grown under moderate (26 L/kg) or severe stress (73 L/kg) by supporting colonized plants in producing higher yield per unit volume of water consumed by the crop in comparison to non-colonized plants under the same level of moderate (43 L/kg) or severe (81 L/kg) water stress. Furthermore, P. indica increased the indole-3-acetic acid (IAA) content, activity levels of catalase (CAT) and peroxidase (POD) with an apparent clear reduction in the abscisic acid (ABA), ethylene, malondialdehyde (MDA), proline contents and stomatal closure compared to non-stressed plants under both water-stress levels. In addition, chlorophyll a, b, a + b contents were increased in the leaves of the colonized plants under water-stress conditions. This improvement in chlorophyll content could be correlated with a significant increment in the transcripts of chlorophyll biosynthesis genes (protochlorophyllide oxidoreductase [POR], chlorophyll a oxygenase [CAO]) and a reduction in the chlorophyll degradation genes (PPH, pheophorbide a oxygenase [PAO], and red chlorophyll catabolite reductase [RCCR]). In conclusion, P. indica has the potential to enhance the cucumber yield grown under moderate water stress rather than severe water stress by improving WUE and altering the activity levels of antioxidant enzymes and chlorophyll metabolism-related genes.

The roles of a light-dependent protochlorophyllide oxidoreductase (LPOR), and ATP-dependent dark operative protochlorophyllide oxidoreductase (DPOR) in chlorophyll biosynthesis

Chlorophyll is a green photosynthetic pigment, and photosynthesis drives the global carbon cycle. The reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) in the penultimate stage of biosynthesis of chlorophyll (Chl) is catalyzed by light-independent protochlorophyllide reducatse (DPOR), and the light-dependent protochlorophyllide oxidoreductase (LPOR). The search was done to all manuscript sections according to terms chlorophyll, a light-dependent protochlorophyllide oxidoreductase, ATP-dependent dark operative protochlorophyllide oxidoreductase, chlorophyll, photosynthesis and chlorophyllide. Within the framework of photosynthesis and chlorophyll, this review article was aimed to provide an overview of the functional studies in chlorophyll biosynthesis, protein crystal structure, disclosure of action mechanisms, and possible future available direction of LPOR and DPOR in the biosynthesis of chlorophyll.

Long-term abscisic acid promotes golden2-like1 degradation through constitutive photomorphogenic 1 in a light intensity-dependent manner to suppress chloroplast development.

Abiotic stress, a serious threat to plants, occurs for extended periods in nature. Abscisic acid (ABA) plays a critical role in abiotic stress responses in plants. Therefore, stress responses mediated by ABA have been studied extensively, especially in short-term responses. However, long-term stress responses mediated by ABA remain largely unknown. To elucidate the mechanism by which plants respond to prolonged abiotic stress, we used long-term ABA treatment that activates the signalling against abiotic stress such as dehydration and investigated mechanisms underlying the responses. Long-term ABA treatment activates constitutive photomorphogenic 1 (COP1). Active COP1 mediates the ubiquitination of golden2-like1 (GLK1) for degradation, contributing to lowering expression of photosynthesis-associated genes such as glutamyl-tRNA reductase (HEMA1) and protochlorophyllide oxidoreductase A (PORA), resulting in the suppression of chloroplast development. Moreover, COP1 activation and GLK1 degradation upon long-term ABA treatment depend on light intensity. Additionally, plants with COP1 mutation or exposed to higher light intensity were more sensitive to salt stress. Collectively, our results demonstrate that long-term treatment of ABA leads to activation of COP1 in a light intensity-dependent manner for GLK1 degradation to suppress chloroplast development, which we propose to constitute a mechanism of balancing normal growth and stress responses upon the long-term abiotic stress.

Accelerated Reaction Engineering of Photo(bio)catalytic Reactions through Parallelization with an Open‐Source Photoreactor

AbstractPhotobiocatalysis is an alternative approach in synthesis that has received much attention in the recent years. Due to the youth of the topic, only few reactor systems are commercially available. To allow a parallel parameter‐screening approach as often used in the optimization of biocatalytic processes, a photoreactor was developed that can illuminate up to 24 samples at well‐defined reaction conditions. The device‘s optical features and temperature regulation have been thoroughly characterized and its application was demonstrated in four examples, specifically three photobiocatalytic and one photocatalytic process: (i) Light‐dependent decarboxylation using a photodecarboxylase; (ii) Reduction of protochlorophyllide using a protochlorophyllide oxidoreductase; (iii) Photosynthetic oxygen production performed by cyanobacteria; and (iv) (−)‐Riboflavin‐catalyzed (E/Z)‐isomerization of cinnamic acid derivatives.

Exogenous Application of 5-Aminolevulinic Acid Promotes Coloration and Improves the Quality of Tomato Fruit by Regulating Carotenoid Metabolism.

5-Aminolevulinic acid (ALA) plays an important role in plant growth and development. It can also be used to enhance crop resistance to environmental stresses and improve the color and internal quality of fruits. However, there are limited reports regarding the effects of ALA on tomato fruit color and its regulatory mechanisms. Therefore, in this study, the effects of exogenous ALA on the quality and coloration of tomato fruits were examined. Tomato (Solanum lycopersicum “Yuanwei No. 1”) fruit surfaces were treated with different concentrations of ALA (0, 100, and 200 mg⋅L–1) on the 24th day after fruit setting (mature green fruit stage), and the content of soluble sugar, titratable acid, soluble protein, vitamin C, and total free amino acids, as well as amino acid components, intermediates of lycopene synthetic and metabolic pathways, and ALA metabolic pathway derivatives were determined during fruit ripening. The relative expression levels of genes involved in lycopene synthesis and metabolism and those involved in ALA metabolism were also analyzed. The results indicated that exogenous ALA (200 mg⋅L–1) increased the contents of soluble sugars, soluble proteins, total free amino acids, and vitamin C as well as 11 kinds of amino acid components in tomato fruits and reduced the content of titratable acids, thus improving the quality of tomato fruits harvested 4 days earlier than those of the control plants. In addition, exogenous ALA markedly improved carotenoid biosynthesis by upregulating the gene expression levels of geranylgeranyl diphosphate synthase, phytoene synthase 1, phytoene desaturase, and lycopene β-cyclase. Furthermore, exogenous ALA inhibited chlorophyll synthesis by downregulating the genes expression levels of Mg-chelatase and protochlorophyllide oxidoreductase. These findings suggest that supplementation with 200 mg⋅L–1 ALA not only enhances the nutritional quality and color of the fruit but also promotes early fruit maturation in tomato.

Elucidation of ligand binding and dimerization of NADPH:protochlorophyllide (Pchlide) oxidoreductase from pea (Pisum sativum L.) by structural analysis and simulations.

NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) is a key enzyme of chlorophyll biosynthesis in angiosperms. It is one of few known photoenzymes, which catalyzes the light-activated trans-reduction of the C17-C18 double bond of Pchlide's porphyrin ring. Due to the light requirement, dark-grown angiosperms cannot synthesize chlorophyll. No crystal structure of POR is available, so to improve understanding of the protein's three-dimensional structure, its dimerization, and binding of ligands (both the cofactor NADPH and substrate Pchlide), we computationally investigated the sequence and structural relationships among homologous proteins identified through database searches. The results indicate that α4 and α7 helices of monomers form the interface of POR dimers. On the basis of conserved residues, we predicted 11 functionally important amino acids that play important roles in POR binding to NADPH. Structural comparison of available crystal structures revealed that they participate in formation of binding pockets that accommodate the Pchlide ligand, and that five atoms of the closed tetrapyrrole are involved in non-bonding interactions. However, we detected no clear pattern in the physico-chemical characteristics of the amino acids they interact with. Thus, we hypothesize that interactions of these atoms in the Pchlide porphyrin ring are important to hold the ligand within the POR binding site. Analysis of Pchlide binding in POR by molecular docking and PELE simulations revealed that the orientation of the nicotinamide group is important for Pchlide binding. These findings highlight the complexity of interactions of porphyrin-containing ligands with proteins, and we suggest that fit-inducing processes play important roles in POR-Pchlide interactions.

In vivo functional analysis of the structural domains of FLUORESCENT (FLU).

The control of chlorophyll (Chl) synthesis in angiosperms depends on the light-operating enzyme protochlorophyllide oxidoreductase (POR). The interruption of Chl synthesis during darkness requires suppression of the synthesis of 5-aminolevulinic acid (ALA), the first precursor molecule specific for Chl synthesis. The inactivation of glutamyl-tRNA reductase (GluTR), the first enzyme in tetrapyrrole biosynthesis, accomplished the decreased ALA synthesis by the membrane-bound protein FLUORESCENT (FLU) and prevents overaccumulation of protochlorophyllide (Pchlide) in the dark. We set out to elucidate the molecular mechanism of FLU-mediated inhibition of ALA synthesis, and explored the role of each of the three structural domains of mature FLU, the transmembrane, coiled-coil and tetratricopeptide repeat (TPR) domains, in this process. Efforts to rescue the FLU knock-out mutant with truncated FLU peptides revealed that, on its own, the TPR domain is insufficient to inactivate GluTR, although tight binding of the TPR domain to GluTR was detected. A truncated FLU peptide consisting of transmembrane and TPR domains also failed to inactivate GluTR in the dark. Similarly, suppression of ALA synthesis could not be achieved by combining the coiled-coil and TPR domains. Interaction studies revealed that binding of GluTR and POR to FLU is essential for inhibiting ALA synthesis. These results imply that all three FLU domains are required for the repression of ALA synthesis, in order to avoid the overaccumulation of Pchlide in the dark. Only complete FLU ensures the formation of a membrane-bound ternary complex consisting at least of FLU, GluTR and POR to repress ALA synthesis.

Arabidopsis JMJ17 promotes cotyledon greening during de-etiolation by repressing genes involved in tetrapyrrole biosynthesis in etiolated seedlings.

Arabidopsis histone H3 lysine 4 (H3K4) demethylases play crucial roles in several developmental processes, but their involvement in seedling establishment remain unexplored. Here, we show that Arabidopsis JUMONJI DOMAIN-CONTAINING PROTEIN17 (JMJ17), an H3K4me3 demethylase, is involved in cotyledon greening during seedling establishment. Dark-grown seedlings of jmj17 accumulated a high concentration of protochlorophyllide, an intermediate metabolite in the tetrapyrrole biosynthesis (TPB) pathway that generates chlorophyll (Chl) during photomorphogenesis. Upon light irradiation, jmj17 mutants displayed decreased cotyledon greening and reduced Chl level compared with the wild-type; overexpression of JMJ17 completely rescued the jmj17-5 phenotype. Transcriptomics analysis uncovered that several genes encoding key enzymes involved in TPB were upregulated in etiolated jmj17 seedlings. Consistently, chromatin immunoprecipitation-quantitative PCR revealed elevated H3K4me3 level at the promoters of target genes. Chromatin association of JMJ17 was diminished upon light exposure. Furthermore, JMJ17 interacted with PHYTOCHROME INTERACTING FACTOR1 in the yeast two-hybrid assay. JMJ17 binds directly to gene promoters to demethylate H3K4me3 to suppress PROTOCHLOROPHYLLIDE OXIDOREDUCTASE C expression and TPB in the dark. Light results in de-repression of gene expression to modulate seedling greening during de-etiolation. Our study reveals a new role for histone demethylase JMJ17 in controlling cotyledon greening in etiolated seedlings during the dark-to-light transition.

Retraction Note: Light Harvesting-like Protein 3 Interacts with Phytoene Synthase and Is Necessary for Carotenoid and Chlorophyll Biosynthesis in Rice

Carotenoid biosynthesis is essential for the generation of photosynthetic pigments, phytohormone production, and flower color development. The light harvesting like 3 (LIL3) protein, which belongs to the light-harvesting complex protein family in photosystems, interacts with geranylgeranyl reductase (GGR) and protochlorophyllide oxidoreductase (POR) both of which are known to regulate terpenoid and chlorophyll biosynthesis, respectively, in both rice and Arabidopsis. In our study, a CRISPR-Cas9 generated 4-bp deletion mutant oslil3 showed aberrant chloroplast development, growth defects, low fertility rates and reduced pigment contents. A comparative transcriptomic analysis of oslil3 suggested that differentially expressed genes (DEGs) involved in photosynthesis, cell wall modification, primary and secondary metabolism are differentially regulated in the mutant. Protein-protein interaction assays indicated that LIL3 interacts with phytoene synthase (PSY) and in addition the gene expression of PSY genes are regulated by LIL3. Subcellular localization of LIL3 and PSY suggested that both are thylakoid membrane anchored proteins in the chloroplast. We suggest that LIL3 directly interacts with PSY to regulate carotenoid biosynthesis. This study reveals a new role of LIL3 in regulating pigment biosynthesis through interaction with the rate limiting enzyme PSY in carotenoid biosynthesis in rice presenting it as a putative target for genetic manipulation of pigment biosynthesis pathways in crop plants.

The Role of Membranes and Lipid-Protein Interactions in the Mg-Branch of Tetrapyrrole Biosynthesis.

Chlorophyll (Chl) is essential for photosynthesis and needs to be produced throughout the whole plant life, especially under changing light intensity and stress conditions which may result in the destruction and elimination of these pigments. All steps of the Mg-branch of tetrapyrrole biosynthesis leading to Chl formation are carried out by enzymes associated with plastid membranes. Still the significance of these protein-membrane and protein-lipid interactions in Chl synthesis and chloroplast differentiation are not very well-understood. In this review, we provide an overview on Chl biosynthesis in angiosperms with emphasis on its association with membranes and lipids. Moreover, the last steps of the pathway including the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), the biosynthesis of the isoprenoid phytyl moiety and the esterification of Chlide are also summarized. The unique biochemical and photophysical properties of the light-dependent NADPH:protochlorophyllide oxidoreductase (LPOR) enzyme catalyzing Pchlide photoreduction and located to peculiar tubuloreticular prolamellar body (PLB) membranes of light-deprived tissues of angiosperms and to envelope membranes, as well as to thylakoids (especially grana margins) are also reviewed. Data about the factors influencing tubuloreticular membrane formation within cells, the spectroscopic properties and the in vitro reconstitution of the native LPOR enzyme complexes are also critically discussed.

Characterization and transcriptomic analysis of a novel yellow-green leaf wucai (Brassica campestris L.) germplasm

BackgroundLeaf color mutants are the ideal materials to explore the pathways of chlorophyll (Chl) metabolism, chloroplast development, and photosynthesis system. In this study, a spontaneous yellow-green leaf wucai (Brassica campestris L.) mutant “WY16–13” was identified, which exhibited yellow-green leaf color during its entire growth period. However, current understanding of the molecular mechanism underlying Chl metabolism and chloroplast development of “WY16–13” is limited.ResultsTotal Chl and carotenoid content in WY16–13 was reduced by 60.92 and 58.82%, respectively, as compared with its wild type parental line W16–13. Electron microscopic investigation revealed fewer chloroplasts per cell and looser stroma lamellae in WY16–13 than in W16–13. A comparative transcriptome profiling was performed using leaves from the yellow-green leaf type (WY16–13) and normal green-leaf type (W16–13). A total of 54.12 million (M) (WY16–13) and 56.17 M (W16–13) reads were generated. A total of 40,578 genes were identified from the mapped libraries. We identified 3882 differentially expressed genes (DEGs) in WY16–13 compared with W16–13 (i.e., 1603 upregulated genes and 2279 downregulated genes). According to the Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, these DEGs are involved in porphyrin and Chl metabolism [i.e., chlorophyllase (CLH), heme oxygenase (HO), chlorophyll (ide) b reductase (NYC), and protochlorophyllide oxidoreductase (POR) genes], carbohydrate metabolism, photosynthesis, and carbon fixation in photosynthetic organisms. Moreover, deficiency in Chl biosynthetic intermediates in WY16–13 revealed that the formation of the yellow-green phenotype was related to the disorder of heme metabolism.ConclusionsOur results provide valuable insights into Chl deficiency in the yellow-green leaf mutant and a bioinformatics resource for further functional identification of key allelic genes responsible for differences in Chl content.

Photocatalytic LPOR forms helical lattices that shape membranes for chlorophyll synthesis.

Chlorophyll biosynthesis, crucial to life on Earth, is tightly regulated because its precursors are phototoxic1. In flowering plants, the enzyme light-dependent protochlorophyllide oxidoreductase (LPOR) captures photons to catalyse the penultimate reaction: the reduction of a double bond within protochlorophyllide (Pchlide) to generate chlorophyllide (Chlide)2,3. In darkness, LPOR oligomerizes to facilitate photon energy transfer and catalysis4,5. However, the complete three-dimensional structure of LPOR, the higher-order architecture of LPOR oligomers and the implications of these self-assembled states for catalysis, including how LPOR positions Pchlide and the co-factor NADPH, remain unknown. Here, we report the atomic structure of LPOR assemblies by electron cryo-microscopy. LPOR polymerizes with its substrates into helical filaments around constricted lipid bilayer tubes. Portions of LPOR and Pchlide insert into the outer membrane leaflet, targeting the product, Chlide, to the membrane for the final reaction site of chlorophyll biosynthesis. In addition to its crucial photocatalytic role, we show that in darkness LPOR filaments directly shape membranes into high-curvature tubules with the spectral properties of the prolamellar body, whose light-triggered disassembly provides lipids for thylakoid assembly. Moreover, our structure of the catalytic site challenges previously proposed reaction mechanisms6. Together, our results reveal a new and unexpected synergy between photosynthetic membrane biogenesis and chlorophyll synthesis in plants, orchestrated by LPOR.

Molecular landscape of etioplast inner membranes in higher plants

Etioplasts are photosynthetically inactive plastids that accumulate when light levels are too low for chloroplast maturation. The etioplast inner membrane consists of a paracrystalline tubular lattice and peripheral, disk-shaped membranes, respectively known as the prolamellar body and prothylakoids. These distinct membrane regions are connected into one continuous compartment. To date, no structures of protein complexes in or at etioplast membranes have been reported. Here, we used electron cryo-tomography to explore the molecular membrane landscape of pea and maize etioplasts. Our tomographic reconstructions show that ATP synthase monomers are enriched in the prothylakoids, and plastid ribosomes in the tubular lattice. The entire tubular lattice is covered by regular helical arrays of a membrane-associated protein, which we identified as the 37-kDa enzyme, light-dependent protochlorophyllide oxidoreductase (LPOR). LPOR is the most abundant protein in the etioplast, where it is responsible for chlorophyll biosynthesis, photoprotection and defining the membrane geometry of the prolamellar body. Based on the 9-Å-resolution volume of the subtomogram average, we propose a structural model of membrane-associated LPOR.

RETRACTED ARTICLE: Light Harvesting-like Protein 3 Interacts with Phytoene Synthase and Is Necessary for Carotenoid and Chlorophyll Biosynthesis in Rice

BackgroundCarotenoid biosynthesis is essential for the generation of photosynthetic pigments, phytohormone production, and flower color development. The light harvesting like 3 (LIL3) protein, which belongs to the light-harvesting complex protein family in photosystems, interacts with geranylgeranyl reductase (GGR) and protochlorophyllide oxidoreductase (POR) both of which are known to regulate terpenoid and chlorophyll biosynthesis, respectively, in both rice and Arabidopsis.ResultsIn our study, a CRISPR-Cas9 generated 4-bp deletion mutant oslil3 showed aberrant chloroplast development, growth defects, low fertility rates and reduced pigment contents. A comparative transcriptomic analysis of oslil3 suggested that differentially expressed genes (DEGs) involved in photosynthesis, cell wall modification, primary and secondary metabolism are differentially regulated in the mutant. Protein-protein interaction assays indicated that LIL3 interacts with phytoene synthase (PSY) and in addition the gene expression of PSY genes are regulated by LIL3. Subcellular localization of LIL3 and PSY suggested that both are thylakoid membrane anchored proteins in the chloroplast. We suggest that LIL3 directly interacts with PSY to regulate carotenoid biosynthesis.ConclusionThis study reveals a new role of LIL3 in regulating pigment biosynthesis through interaction with the rate limiting enzyme PSY in carotenoid biosynthesis in rice presenting it as a putative target for genetic manipulation of pigment biosynthesis pathways in crop plants.

Photocatalysis as the 'master switch' of photomorphogenesis in early plant development.

Enzymatic photocatalysis is seldom used in biology. Photocatalysis by light-dependent protochlorophyllide oxidoreductase (LPOR)-one of only a few natural light-dependent enzymes-is an exception, and is responsible for the conversion of protochlorophyllide to chlorophyllide in chlorophyll biosynthesis. Photocatalysis by LPOR not only regulates the biosynthesis of the most abundant pigment on Earth but it is also a 'master switch' in photomorphogenesis in early plant development. Following illumination, LPOR promotes chlorophyll production, plastid membranes are transformed and the photosynthetic apparatus is established. Given these remarkable, light-induced pigment and morphological changes, the LPOR-catalysed reaction has been extensively studied from catalytic, physiological and plant development perspectives, highlighting vital, and multiple, cellular roles of this intriguing enzyme. Here, we offer a perspective in which the link between LPOR photocatalysis and plant photomorphogenesis is explored. Notable breakthroughs in LPOR structural biology have uncovered the structural-mechanistic basis of photocatalysis. These studies have clarified how photon absorption by the pigment protochlorophyllide-bound in a ternary LPOR-protochlorophyllide-NADPH complex-triggers photocatalysis and a cascade of complex molecular and cellular events that lead to plant morphological changes. Photocatalysis is therefore the master switch responsible for early-stage plant development and ultimately life on Earth.

An RNA-seq Analysis Reveals Differential Transcriptional Responses to Different Light Qualities in Leaf Color of Camellia sinensis cv. Huangjinya

‘Huangjinya’ is a novel light-sensitive albino tea cultivar. Previously we have found that the leaves exhibit varying degrees of yellow coloration after being exposed to different light qualities, including red light (RL), blue light (BL), red + blue light (RB) and white light (WL). To understand the transcriptional response of different light qualities in leaf coloration of ‘Huangjinya’, RNA-seq analysis with three biological replicates was performed to identify differentially expressed genes (DEGs) involved in pigment metabolism pathways. With regard to porphyrin and chlorophyll metabolism, the protochlorophyllide oxidoreductase (POR) was upregulated and chlorophyllase (Chlase) was downregulated under RL compared with WL, which contributed to high chlorophyll content in ‘Huangjinya’. The upregulated POR and magnesium chelatase H subunit (CHLH) were identified under RB. BL significantly promoted carotenoid biosynthesis, accompanied by the related upregulated genes. However, upregulated xanthoxin dehydrogenase (ABA2) reduced carotenoid content under BL. Zeaxanthin epoxidase (ZEP) was upregulated and ABA2 was downregulated under RB, resulting in high accumulation of carotenoid content. BL and RL upregulated expressions of genes involved in flavonoid biosynthesis in ‘Huangjinya’. Differential expressions of genes involved in flavonoid biosynthesis was considered as the results of leaf color change. In addition, the genes related to photosynthesis were downregulated under RL, whereas these were upregulated under BL when compared with WL. In conclusion, the effect of light quality on the leaf coloration of ‘Huangjinya’ was mainly dependent on chlorophyll content by altering corresponding genes expressions. RL may promote the accumulation of chlorophyll in ‘Huangjinya’ leaves by upregulating the expression of genes related to chlorophyll synthesis (e.g., POR) and downregulating expression of genes related to chlorophyll degradation (e.g., Chlase). BL could upregulate the expression of genes related to both biosynthesis (e.g., POR, hemA) and degradation (e.g., Chlase) of chlorophyll, resulting in little change in leaf color of ‘Huangjinya’ under blue light when compared with white light. Upregulated genes involved in chlorophyll biosynthesis (e.g., POR, CHLH) induced greener color of ‘Huangjinya’ treated with RB.

The flexible N-terminus of BchL autoinhibits activity through interaction with its [4Fe-4S] cluster and released upon ATP binding

A key step in bacteriochlorophyll biosynthesis is the reduction of protochlorophyllide to chlorophyllide, catalyzed by dark-operative protochlorophyllide oxidoreductase. Dark-operative protochlorophyllide oxidoreductase contains two [4Fe-4S]–containing component proteins (BchL and BchNB) that assemble upon ATP binding to BchL to coordinate electron transfer and protochlorophyllide reduction. But the precise nature of the ATP-induced conformational changes is poorly understood. We present a crystal structure of BchL in the nucleotide-free form where a conserved, flexible region in the N-terminus masks the [4Fe-4S] cluster at the docking interface between BchL and BchNB. Amino acid substitutions in this region produce a hyperactive enzyme complex, suggesting a role for the N-terminus in autoinhibition. Hydrogen–deuterium exchange mass spectrometry shows that ATP binding to BchL produces specific conformational changes leading to release of the flexible N-terminus from the docking interface. The release also promotes changes within the local environment surrounding the [4Fe-4S] cluster and promotes BchL-complex formation with BchNB. A key patch of amino acids, Asp-Phe-Asp (the ‘DFD patch’), situated at the mouth of the BchL ATP-binding pocket promotes intersubunit cross stabilization of the two subunits. A linked BchL dimer with one defective ATP-binding site does not support protochlorophyllide reduction, illustrating nucleotide binding to both subunits as a prerequisite for the intersubunit cross stabilization. The masking of the [4Fe-4S] cluster by the flexible N-terminal region and the associated inhibition of the activity is a novel mechanism of regulation in metalloproteins. Such mechanisms are possibly an adaptation to the anaerobic nature of eubacterial cells with poor tolerance for oxygen.

Methods for Heterologous Overproduction of Fe-S Proteins.

Proteins carrying iron-sulfur ([Fe-S]) clusters are critical to the basic metabolism of all organisms. Structural and biochemical investigations of many such [Fe-S] cluster proteins depend on recombinant overproduction using heterologous bacterial hosts such as Escherichia coli . Here, we describe a detailed procedure for the overproduction and purification of two oxygen-sensitive component proteins of the dark-operative protochlorophyllide oxidoreductase (DPOR) complex. The method relies on an engineered Escherichia coli cell line carrying a correction in its genome to restore the loss of a key [Fe-S] cluster biogenesis pathway. The method can also be potentially adapted for the overproduction of other Fe-S proteins.

Complex Evolution of Light-Dependent Protochlorophyllide Oxidoreductases in Aerobic Anoxygenic Phototrophs: Origin, Phylogeny, and Function.

Light-dependent protochlorophyllide oxidoreductase (LPOR) and dark-operative protochlorophyllide oxidoreductase are evolutionary and structurally distinct enzymes that are essential for the synthesis of (bacterio)chlorophyll, the primary pigment needed for both anoxygenic and oxygenic photosynthesis. In contrast to the long-held hypothesis that LPORs are only present in oxygenic phototrophs, we recently identified a functional LPOR in the aerobic anoxygenic phototrophic bacterium (AAPB) Dinoroseobacter shibae and attributed its presence to a single horizontal gene transfer event from cyanobacteria. Here, we provide evidence for the more widespread presence of genuine LPOR enzymes in AAPBs. An exhaustive bioinformatics search identified 36 putative LPORs outside of oxygenic phototrophic bacteria (cyanobacteria) with the majority being AAPBs. Using in vitro and in vivo assays, we show that the large majority of the tested AAPB enzymes are genuine LPORs. Solution structural analyses, performed for two of the AAPB LPORs, revealed a globally conserved structure when compared with a well-characterized cyanobacterial LPOR. Phylogenetic analyses suggest that LPORs were transferred not only from cyanobacteria but also subsequently between proteobacteria and from proteobacteria to Gemmatimonadetes. Our study thus provides another interesting example for the complex evolutionary processes that govern the evolution of bacteria, involving multiple horizontal gene transfer events that likely occurred at different time points and involved different donors.