Identification of NADPH:protochlorophyllide oxidoreductases A and B: a branched pathway for light-dependent chlorophyll biosynthesis in Arabidopsis thaliana.

Illumination releases the arrest in chlorophyll (Chl) biosynthesis in etiolated angiosperm seedlings through the enzymatic photoreduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), the first light-dependent step in chloroplast biogenesis. NADPH: Pchlide oxidoreductase (POR, EC 1.3.1.33), a nuclear-encoded plastid-localized enzyme, mediates this unique photoreduction. Paradoxically, light also triggers a drastic decrease in the amounts of POR activity and protein before the Chl accumulation rate reaches its maximum during greening. While investigating this seeming contradiction, we identified two distinct Arabidopsis thaliana genes encoding POR, in contrast to previous reports of only one gene in angiosperms. The genes, designated PorA and PorB, by analogy to the principal members of the phytochrome photoreceptor gene family, display dramatically different patterns of light and developmental regulation. PorA mRNA disappears within the first 4 h of greening, whereas PorB mRNA persists even after 16 h of illumination, mirroring the behavior of two distinct POR protein species. Experiments designed to help define the functions of POR A and POR B demonstrate exclusive expression of PorA in young seedlings and of PorB both in seedlings and in adult plants. Accordingly, we propose the existence of a branched light-dependent Chl biosynthesis pathway in which POR A performs a specialized function restricted to the initial stages of greening and POR B maintains Chl levels throughout angiosperm development.     

Regulation of etioplast pigment-protein complexes, inner membrane architecture, and protochlorophyllide a chemical heterogeneity by light-dependent NADPH:protochlorophyllide oxidoreductases A and B

The etioplast of dark-grown angiosperms is characterized by the prolamellar body (PLB) inner membrane, the absence of chlorophyll, and the accumulation of divinyl and monovinyl derivatives of protochlorophyll(ide) a [Pchl(ide) a]. Either of two structurally related, but differentially expressed light-dependent NADPH:Pchlide oxidoreductases (PORs), PORA and PORB, can assemble the PLB and form dark-stable ternary complexes containing enzymatically photoactive Pchlide-F655. Here we have examined in detail whether these polypeptides play redundant roles in etioplast differentiation by manipulating the total POR content and the PORA-to-PORB ratio of etiolated Arabidopsis seedlings using antisense and overexpression approaches. POR content correlates closely with PLB formation, the amounts, spectroscopic properties, and photoreduction kinetics of photoactive Pchlide, the ratio of photoactive Pchlide-F655 to non-photoactive Pchl(ide)-F632, and the ratio of divinyl- to monovinyl-Pchl(ide). This last result defines POR as the first endogenous protein factor demonstrated to influence the chemical heterogeneity of Pchl(ide) in angiosperms. It is intriguing that excitation energy transfer between different spectroscopic forms of Pchl(ide) in etiolated cotyledons remains largely independent of POR content. We therefore propose that the PLB contains a minimal structural unit with defined pigment stoichiometries, within which a small amount of non-photoactive Pchl(ide) transfers excitation energy to a large excess of photoactive Pchlide-F655. In addition, our data suggests that POR may bind not only stoichiometric amounts of photoactive Pchlide, but also substoichiometric amounts of non-photoactive Pchl(ide). We conclude that the typical characteristics of etioplasts are closely related to total POR content, but not obviously to the specific presence of PORA or PORB.     

A role of Toc33 in the protochlorophyllide-dependent plastid import pathway of NADPH:protochlorophyllide oxidoreductase (POR) A

NADPH:protochlorophyllide oxidoreductase (POR) A is a key enzyme of chlorophyll biosynthesis in angiosperms. It is nucleus-encoded, synthesized as a larger precursor in the cytosol and imported into the plastids in a substrate-dependent manner. Plastid envelope membrane proteins, called protochlorophyllide-dependent translocon proteins, Ptcs, have been identified that interact with pPORA during import. Among them are a 16-kDa ortholog of the previously characterized outer envelope protein Oep16 (named Ptc16) and a 33-kDa protein (Ptc33) related to the GTP-binding proteins Toc33 and Toc34 of Arabidopsis. In the present work, we studied the interactions and roles of Ptc16 and Ptc33 during pPORA import. Radiolabeled Ptc16/Oep16 was synthesized from a corresponding cDNA and imported into isolated Arabidopsis plastids. Crosslinking experiments revealed that import of 35S-Oep16/Ptc16 is stimulated by GTP. 35S-Oep16/Ptc16 forms larger complexes with Toc33 but not Toc34. Plastids of the ppi1 mutant of Arabidopsis lacking Toc33, were unable to import pPORA in darkness but imported the small subunit precursor of ribulose-1,5-bisphosphate carboxylase/oxygenase (pSSU), precursor ferredoxin (pFd) as well as pPORB which is a close relative of pPORA. In white light, partial suppressions of pSSU, pFd and pPORB import were observed. Our results unveil a hitherto unrecognized role of Toc33 in pPORA import and suggest photooxidative membrane damage, induced by excess Pchlide accumulating in ppi1 chloroplasts because of the lack of pPORA import, to be the cause of the general drop of protein import.     

Arabidopsis light-dependent NADPH: protochlorophyllide oxidoreductase A (PORA) is essential for normal plant growth and development: an addendum

Recently the porA-1 null mutant of Arabidopsis thaliana has been identified, which contains an insertion of the Dissociation (Ds) element in the PORA gene (Paddock et al. in Plant Mol Biol 78:447-460, 2012). Light-grown porA-1 seedlings suffer from a drastically reduced chlorophyll content and a developmental arrest beyond the cotyledon stage, suggesting that PORA is not only transiently involved in initiating chlorophyll synthesis during illumination of etiolated seedlings but is also essential for normal growth and plant development. Here we report the presence of a second Ds element in this porA-1 mutant line that inactivates the Speechless gene required for stomata formation. Similar to porA-1, speechless seedlings are severely impaired in their development. Our results suggest that the lack of stomata in porA-1 may contribute to the dwarfed phenotype of the mutant and thus emphasizes the need to re-address the proposed role of PORA during plant development by studying a porA mutant that retains its stomata formation.     

Association of the NADPH:protochlorophyllide oxidoreductase (POR) with isolated etioplast inner membranes from wheat

Membrane association of NADPH:protochlorophyllide oxidoreductase (POR, EC: 1.6.99.1) with isolated prolamellar bodies (PLBs) and prothylakoids (PTs) from wheat etioplasts was investigated. In vitro-expressed radiolabelled POR, with or without transit peptide, was used to characterize membrane association conditions. Proper association of POR with PLBs and PTs did not require the presequence, whereas NADPH and hydrolysable ATP were vital for the process. After treating the membranes with thermolysin, sodium hydroxide or carbonate, a firm attachment of the POR protein to the membrane was found. Although the PLBs and PTs differ significantly in their relative amount of POR in vivo, no major differences in POR association capacity could be observed between the two membrane systems when exogenous NADPH was added. Experiments run with only an endogenous NADPH source almost abolished association of POR with both PLBs and PTs. In addition, POR protein carrying a mutation in the putative nucleotide-binding site (ALA06) was unable to bind to the inner membranes in the presence of NADPH, which further demonstrates that the co-factor is essential for proper membrane association. POR protein carrying a mutation in the substrate-binding site (ALA24) showed less binding to the membranes as compared to the wild type. The results presented here introduce studies of a novel area of protein-membrane interaction, namely the association of proteins with a paracrystalline membrane structure, the PLB.     

NADPH : protochlorophyllide oxidoreductases in white pine (Pines strobes) and loblolly pine (P. taeda)

Summary NADPH : protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) catalyzes the light-dependent reduction of protochlorophyllide in higher plants. Cloned cDNAs encoding two distinct pchlide reductases were isolated from a gt11 library constructed from poly(A)⁺ RNA prepared from the cotyledons of dark-grown white pine (Pines strobes) seedlings and a nuclear gene (lpcr) analogous to one of these cDNAs has been characterized from loblolly pine (P. taeda). The pine gene encodes an approximately 43 kDa precursor polypeptide consisting of a 334-amino acid mature protein and a 66-amino acid transit peptide. The deduced primary structures for the pine proteins are highly homologous to those reported from monocots and dicots. The coding portion of the pine lpcr gene is interrupted by four introns. The placement of these introns within the pine lpcr gene is identical to that observed in pea (Pisum sativum), suggesting conservation in gene organization between dicot and gymnosperm species. Western blot analysis using polyclonal antiserum against oat pchlide reductase detected in extracts of dark-grown pine cotyledons a single immunoreactive protein, which declined in abundance during a 48 h period of illumination with white light. Cotyledons of dark-grown seedlings were also found to accumulate high levels of pchlide reductase mRNA; however, little or no change in the steady-state levels of mRNA encoding pchlide reductase was observed in these tissues following illumination. Stem tissue of dark-grown seedlings did not contain significant levels of pchlide reductase mRNA, whereas stems of light-grown plants of the same age accumulated substantial amounts of the message. These results suggest that light and the developmental age of the tissue affect regulation of lpcr expression in pine.     

Functional analysis of isoforms of NADPH: protochlorophyllide oxidoreductase (POR), PORB and PORC, in Arabidopsis thaliana

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide. To elucidate the physiological function of three differentially regulated POR isoforms (PORA, PORB and PORC) in Arabidopsis thaliana, we isolated T-DNA tagged null mutants of porB and porC. The mature seedlings of the mutants had normal photosynthetic competencies, showing that PORB and PORC are interchangeable and functionally redundant in developed plants. In etiolated seedlings, only porB showed a reduction in the photoactive protochlorophyllide and the size of prolamellar bodies (PLBs), indicating that PORB, as well as PORA, functioned in PLB assembly and photoactive protochlorophyllide formation in etiolated seedlings. When illuminated, the etiolated porB seedling was able to green to a similar extent as the wild type, whereas the greening was significantly reduced under low light conditions. During greening, high light irradiation increased the level of PORC protein, and the greening of porC was repressed under high light conditions. The porB, but not porC, etiolated seedling was more sensitive to the far-red block of greening than the wild type, which is caused by depletion of endogenous POR proteins resulting in photo-oxidative damage. These results suggest that, at the onset of greening, PLBs are important for efficient capture of light energy for photoconversion under various light conditions, and PORC, which is induced by high light irradiation, contributes to photoprotection during greening of the etiolated seedlings.     

Assembly of NADPH:protochlorophyllide oxidoreductase complex is needed for effective greening of barley seedlings

NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) is the key enzyme in the light-induced greening of higher plants. A unique light-harvesting POR:Pchlide complexes (LHPP) has been found in barley etioplasts, but not in other plant species. Why PORs from barley, but not from other plants, can form LHPP? And its function is not well understood. We modeled the barley and Arabidopsis POR proteins and compared molecular surface. The results confirm the idea that barley PORA can form a five-unit oligomer that interacts with a single PORB. Chemical treatment experiments indicated that POR complex may be formed by dithiol oxidation of cysteines of two adjacent proteins. We further showed that LHPP assembly was needed for barley POR functions and seedling greening. On the contrary, Arabidopsis POR proteins only formed dimers, which were not related to the functions or the greening. Finally, POR complex assembly (including LHPP and POR dimers) did not affect the formation of prolamellar bodies (PLBs) that function for efficient capture of light energy for photo conversion in etioplasts.     

Arabidopsis light-dependent protochlorophyllide oxidoreductase A (PORA) is essential for normal plant growth and development

During skotomorphogenesis in angiosperms, NADPH:protochlorophyllide oxidoreductase (POR) forms an aggregate of photolabile NADPH-POR-protochlorophyllide (Pchlide) ternary complexes localized to the prolamellar bodies within etioplasts. During photomorphogenesis, POR catalyzes the light-dependent reduction of Pchlide a to chlorophyllide (Chlide) a, which is subsequently converted to chlorophyll (Chl). In Arabidopsis there are three structurally related POR genes, denoted PORA, PORB and PORC. The PORA and PORB proteins accumulate during skotomorphogenesis. During illumination, PORA is only transiently expressed, whereas PORB and PORC persist and are responsible for bulk Chl synthesis throughout plant development. Here we have tested whether PORA is important for skotomorphogenesis by assisting in etioplast development, and normal photomorphogenic development. Using reverse genetic approaches, we have identified the porA-1 null mutant, which contains an insertion of the maize Dissociation transposable element in the PORA gene. Additionally, we have characterized PORA RNAi lines. The porA-1 and PORA RNAi lines display severe photoautotrophic growth defects, which can be partially rescued on sucrose-supplemented growth media. Elimination of PORA during skotomorphogenesis results in reductions in the volume and frequency of prolamellar bodies, and in photoactive Pchlide conversion. The porA-1 mutant characterization thus establishes a quantitative requirement for PORA in etioplast development by demonstrating significant membrane ultrastructural and biochemical defects, in addition to suggesting PORA-specific functions in photomorphogenesis and plant development.     

Origin and evolution of the light-dependent protochlorophyllide oxidoreductase (LPOR) genes

Abstract: Light‐dependent NADPH‐protochlorophyllide oxidoreductase (LPOR) is a nuclear‐encoded chloroplast protein in green algae and higher plants which catalyzes the light‐dependent reduction of protochlorophyllide to chlorophyllide. Light‐dependent chlorophyll biosynthesis occurs in all oxygenic photosynthetic organisms. With the exception of angiosperms, this pathway coexists with a separate light‐independent chlorophyll biosynthetic pathway, which is catalyzed by light‐independent protochlorophyllide reductase (DPOR) in the dark. In contrast, the light‐dependent function of chlorophyll biosynthesis is absent from anoxygenic photosynthetic bacteria. Consequently, the question is whether cyanobacteria are the ancestors of all organisms that conduct light‐dependent chlorophyll biosynthesis. If so, how did photosynthetic eukaryotes acquire the homologous genes of LPOR in their nuclear genomes? The large number of complete genome sequences now available allow us to detect the evolutionary history of LPOR genes by conducting a genome‐wide sequence comparison and phylogenetic analysis. Here, we show the results of a detailed phylogenetic analysis of LPOR and other functionally related enzymes in the short chain dehydrogenase/reductase (SDR) family. We propose that the LPOR gene originated in the cyanobacterial genome before the divergence of eukaryotic photosynthetic organisms. We postulated that the photosynthetic eukaryotes obtained their LPOR homologues through endosymbiotic gene transfer.     

The light-dependent regulation of gene expression during plastid development in higher plants

In recent years there has been a considerable increase in our understanding of the manner by which light affects gene expression during chloroplast development. In most systems that have been studied, light acts through sensitive photoreceptor molecules and quantitatively increases or represses the level of expression of specific nuclear-and plastid-encoded genes. Although the mechanisms are obscure, a picture is beginning to emerge in which the coordination of nuclear and plastid gene expression is controlled by regulatory mechanisms originating within their respective subcellular compartments. This review summarizes some of our current knowledge concerning the nature of light-regulated gene expression in higher plants and provides a prospectus for future research in this area.     

Regeneration of protochlorophyllide in green and greening leaves of plants with varying proportions of protochlorophyllide forms in darkness

During illumination of dark-grown plants protochlorophyllide (Pchlide) is continuously transformed to chlorophyllide (Chlide). Different dark-grown plants, maize ( Zea mays cv. Sundance), wheat ( Triticum aestivum cv. Kosack), pea ( Pisum sativum cv. Kelwedon wonder), the lip1 mutant of pea, and the aurea mutant of tomato ( Solanum lycopersicum ), have various ratios of spectral Pchlide forms in darkness. When the plants were illuminated and then returned to darkness Pchlide re-accumulated. The proportions of different Pchlide forms within the pool of re-accumulated Pchlide were followed by low temperature fluorescence emission and excitation spectra in green and greening leaves. After 1 h of illumination the spectral characteristics of regenerated Pchlide forms mirrored those of Pchlide in dark-grown plants and were thus species dependent. After a prolonged illumination period (24 h) as well as in fully green leaves energy transfer to chlorophyll (Chl) masked the presence of long-wavelength Pchlide in the fluorescence emission spectra. However, excitation spectra showed Pchlide absorption around 650 nm and its flash-induced disappearance confirmed its nature of phototransformable Pchlide. In fact the excitation spectra showed that the proportions of different Pchlide forms in green leaves highly resembled the proportions of Pchlide forms in dark-grown leaves and were specific for the plant variety. Thus Chl formation in both dark-grown and light-grown leaves can occur in a similar way through the main photoactive long-wavelength form of Pchlide.     

In vitro-mutagenesis of NADPH:protochlorophyllide oxidoreductase B: two distinctive protochlorophyllide binding sites participate in enzyme catalysis and assembly

NADPH:protochlorophyllide oxidoreductase (POR) B is a key enzyme for the light-induced greening of etiolated angiosperm plants. It is nucleus-encoded, imported into the plastids posttranslationally, and assembled into larger light-harvesting POR:protochlorophyllide complexes termed LHPP (Reinbothe et al., Nature 397:80–84, 1999). An in vitro-mutagenesis approach was taken to study the role of the evolutionarily conserved Cys residues in pigment binding. Four Cys residues are present in the PORB of which two, Cys276 and Cys303, established distinct pigment binding sites, as shown by biochemical tests, protein import studies, and in vitro-reconstitution experiments. While Cys276 constituted the Pchlide binding site in the active site of the enzyme, Cys303 established a second, low affinity pigment binding site that was involved in the assembly and stabilization of imported PORB enzyme inside etioplasts.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

The charge-transfer complex between protochlorophyllide and NADPH: an intermediate in protochlorophyllide photoreduction

A hypothesis describing the mechanism of photoactive protochlorophyllide (P) photoreduction in vivo, relating mainly to the molecular nature of the intermediates, is proposed. The hypothesis is compatible with currently published experimental data. After illumination of etiolated barley leaves at 143 to 153 K, the absorption of P remains essentially unchanged, but a new absorption band at 690 nm is observed. Appearance of this new intermediate enables to distinguish between light and dark stages of the photoconversion reaction. When returned to the higher temperature in the dark, the treated leaves begin accumulating chlorophyllide (Chlide), concomitant with the disappearance of the 690-nm band. The decay time of the excited P (P^*) is estimated at 300 ps, which approximates the time constant of photoinduced electron transfer (ET). It is suggested that the charge-transfer complex (CTC) in its ground state (GS) (ground state of CTC formed by the partial (δ) electron transfer), i.e. (P^δ−•••H–D^δ+), between P and NADPH – the electron and proton donor (H–D) – accumulates in the following sequence: P^* + H–D → (P^*•••H–D)→[(P^*•••H–D)←(P⁻•••H–D⁺)] → ¹(P⁻•••H–D⁺)] → ³(P⁻•••H–D⁺) → (P^δ−•••H–D ^δ+), where an equilibrium state (ES) – [(P^*•••H–D)←(P⁻•••H–D⁺)] – with a lifetime of about 1 to 2 ns, exists between the local excited (LE) and ET states. The existence of a triplet ET state – ³(P⁻•••H–D⁺) – is proposed because the time interval between recording of the ES and appearance of the CTC GS (35–250 ns) does not fit the lifetime of the singlet excited complex (exciplex). It is feasible that apart from NADPH, other intermediate proton carriers are contemporaneously involved in the dark reaction (P^δ−•••H–D^δ+) → Chlide, because proton binding to the C₇–C₈ bond in vivo takes place in the trans-configuration. The hydride ion may approach the C₇–C₈ bond from one side by heterolytic fission and an additional proton, donated by the protein group, may be simultaneously added to this bond from the opposite side of the porphyrin nucleus surface. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mercury Inhibits the Activity of the NADPH:Protochlorophyllide Oxidoreductase (POR)

The effect of Hg⁺⁺ was studied on the arrangement and photoactivity of NADPH:protochlorophyllide oxidoreductase (POR) in homogenates of dark-grown wheat (Triticum aestivum L.) leaves. 77 K fluorescence emission spectra of the homogenates were recorded before and after the irradiation of the homogenates and the spectra were deconvoluted into Gaussian components. The mercury treatment caused a precipitation of the membrane particles, which was followed by a remarkable decrease of the fluorescence yield. 10^-3 M Hg⁺⁺ decreased the ratio of the 655 nm-emitting protochlorophyllide (Pchlide) form to the 633 nm-emitting form. 10^-2 M Hg⁺⁺ shifted the short wavelength band to 629–630 nm and a 655 nm form was observed which was inactive on irradiation. This inhibition may be caused by serious alteration of the enzyme structure resulting in the trans-localisation of NADPH within the active site of POR.     

Monovinyl and divinyl protochlorophyllide pools in etiolated tissues of higher plants

The composition of chlorophyll-precursor pigments, particularly the contents of monovinyl (MV) and divinyl (DV) protochlorophyllides (Pchlides), in etiolated tissues of higher plants were determined by polyethylene-column HPLC (Y. Shioi, S. I. Beale [1987] Anal Biochem 162: 493-499), which enables the complete separation of these pigments. DV-Pchlide was ubiquitous in etiolated tissue of higher plants. From the analyses of 24 plant species belonging to 17 different families, it was shown that the concentration of DV-Pchlide was strongly dependent on the plant species and the age of the plants. The ratio of DV-Pchlide to MV-Pchlide in high DV-Pchlide plants such as cucumber and leaf mustard decreased sharply with increasing age. Levels of DV-Pchlide in Gramineae plants were considerably lower at all ages compared with those of other plants. Etiolated tissues of higher plants such as barley and corn were, therefore, good sources of MV-Pchlide. Absorption spectra of the purified MV- and DV-Pchlides in ether are presented and compared.     

Characterisation of the assembly pathway of the pea NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR), with emphasis on the role of its substrate, Pchlide

The homologous import and membrane association of a key enzyme for chlorophyll biosynthesis, the NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR, EC 1.6.99.1) into pea chloroplasts was investigated in vitro. The co-factor, NADPH, decreased binding of the precursor protein (pPOR) to the envelope membranes in the presence of ATP. The decrease of the binding reaction with NADPH was not observed with the precursor of the small subunit of Rubisco (pSS).
To investigate possible substrate-dependency for the import reaction, internal Pchlide concentrations in the plastids were raised by either an addition of δ -aminolevulinic acid to isolated plastids or etiolation of the seedlings prior to plastid isolation. Increased amounts of plastid-bound Pchlide gave no observable differences in POR import.
The capacity of POR and 11 different POR mutants, carrying charged-to-alanine scanning substitutions, to form a catalytically active POR-Pchlide-NADPH complex and to associate with the thylakoid membranes in a protease-resistant way were tested. Wild-type POR, as well as the mutants with charge substitutions in the N-terminal region of the protein, exhibited higher catalytic activity than the POR mutants carrying substitutions in the C-terminal region. Formation of a catalytically active complex did not, however, increase the association efficiency onto the thylakoids. We can, therefore, postulate that the import of pea POR into pea chloroplasts was not substrate-dependent, nor did formation of catalytically active complexes stimulate or inhibit the membrane association reaction of POR.