Beata Mysliwa-Kurdziel

Light-Dependent Protochlorophyllide Oxidoreductase: Phylogeny, Regulation, and Catalytic Properties

This Current Topic focuses on light-dependent protochlorophyllide oxidoreductase (POR, EC 1.3.1.33). POR catalyzes the penultimate reaction of chlorophyll biosynthesis, i.e., the light-triggered reduction of protochlorophyllide to chlorophyllide. In this reaction, the chlorin ring of the chlorophyll molecule is formed, which is crucial for photosynthesis. POR is one of very few enzymes that are driven by light; however, it is unique in the need for its substrate to absorb photons to induce the conformational changes in the enzyme, which are required for its catalytic activation. Moreover, the enzyme is also involved in the negative feedback of the chlorophyll biosynthesis pathway and controls chlorophyll content via its light-dependent activity. Even though it has been almost 70 years since the first isolation of active POR complexes, our knowledge of them has markedly advanced in recent years. In this review, we summarize the current state of knowledge of POR, including the phylogenetic roots of POR, the mechanisms of the regulation of POR genes expression, the regulation of POR activity, the import of POR into plastids, the role of POR in PLB formation, and the molecular mechanism of protochlorophyllide reduction by POR. To the best of our knowledge, no previous review has compiled such a broad set of recent findings about POR.

Analysis of fluorescence lifetime of protochlorophyllide and chlorophyllide in isolated etioplast membranes measured from multifrequency cross-correlation phase fluorometry

The fluorescence decays of protochlorophyllide (Pchlide) and of chlorophyllide (Chlide) in wheat etioplast membranes were analyzed using a multiexponential fluorescence decay model. Using different excitation wavelengths from 430 to 470 nm, we found that a triple‐exponential model at 14°C and a double‐exponential model at — 170°C were adequate to describe the Pchlide fluorescence decay. We discuss the origin of the three fluorescence lifetime components at 14°C on the basis of the dependence of their fractional intensities on the excitation wavelength and by correlating the fractional intensities with integrated fluorescence intensities of different Pchlide forms in steady‐state fluorescence spectra. The fluorescence decay of the main Pchlide form, photoactive Pchlide‐F657, is shown to have a complex character with a fast component of 0.25 ns and a slower component of about 2 ns. Two lifetime components of 2 ns and 5.5–6.0 ns are ascribed to the second photoactive form, Pchlide‐F645, and to nonphotoactive Pchlide forms, respectively. In etioplast membranes preilluminated by a short saturating light pulse, we found a single 5.0 ns component for Chlide‐F688 (the Chlide‐NADPH: protochlorophyllide oxidoreductase [PORJ‐NADP+complex) and an additional 1.6 ns component when the formation of Chlide‐F696 (the Chlide‐POR‐NADPH complex) was promoted by exogenous NADPH. From the fluorescence lifetime results we evaluated the quantum yield of the primary photoreaction by Chlide‐F696 as being 70%.

Photoactive Protochlorophyllide-Enzyme Complexes Reconstituted with PORA, PORB and PORC Proteins of A. thaliana : Fluorescence and Catalytic Properties

Photoactive Pchlide-POR-NADPH complexes were reconstituted using protochlorophyllide (Pchlide) and recombinant light-dependent protochlorophyllide oxidoreductase (POR) proteins, His₆-PORA, His₆-PORB and His₆-PORC, from Arabidopsis thaliana. We did not observe any differences in the kinetics of the protochlorophyllide photoreduction at room temperature among the PORA, PORB and PORC proteins. In contrast, the PORC protein showed lower yield of Chlide formation than PORA and PORB when preincubated in the dark for 30 min and then illuminated for a short time. The most significant observation was that reconstituted Pchlide-POR-NADPH complexes showed fluorescence maxima at 77 K similar to those observed for highly aggregated Pchlide-POR-NADPH complexes in prolamellar bodies (PLBs) in vivo. Homology models of PORA, PORB and PORC of Arabidopsis thaliana were developed to compare predicted structures of POR isoforms. There were only slight structural differences, mainly in the organisation of helices and loops, but not in the shape of whole molecules. This is the first comparative analysis of all POR isoforms functioning at different stages of A. thaliana development.

Light-dependent and light-independent protochlorophyllide oxidoreductases share similar sequence motifs —in silico studies

In the present studies, we have found a fragment of amino acid sequence, called TFT motif, both in light-dependent protochlorophyllide oxidoreductase (LPOR) and in the L subunit of dark-operative (light-independent) protochlorophyllide oxidoreductases (DPOR). Amino acid residues of this motif shared similar physicochemical properties in both types of the enzymes. In the present paper, physicochemical properties of amino acid residues of this common motif, its spatial arrangement and a possible physiological role are being discussed. This is the first report when similarity between LPOR and DPOR, phylogenetically unrelated, but functionally redundant enzymes, is described.

Changes in endothermic transitions associated with light-induced chlorophyllide formation, as investigated by differential scanning calorimetry

The changes in thermal transitions associated with protochlorophyllide (Pchlide) into chlorophyllide (Chlide) phototransformation were investigated using sensitive differential scanning calorimetry (DSC). Two groups of endothermic transitions, each composed of several components, were observed between 25 and 90°C for prolamellar body (PLB) membranes isolated from etiolated wheat (Triticum aestivum) leaves. The first group, located in the 20-45°C region, was strongly affected by a short light pulse given prior to measurement. A decrease of this group of transitions was observed shortly after illumination and it was hardly detected in PLBs measured after the Chlide Shibata shift. Calorimetric results were supported by 77-K fluorescence emission spectra measured for continuously heated samples at different temperatures. We interpreted the DSC band observed between 20 and 45°C as due to the disaggregation of Pchlide- (or Chlide-)-reductase-NADPH complexes. More detailed analysis using Gaussian deconvolution showed that this band was composed of three transitions at 32, 39 and 41°C. The second group of transitions was detected in the 45-80°C region, the same as for mature thylakoids. Its main component at 60°C was not affected significantly by a short light pulse. By comparison of thermograms obtained for PLBs with those for mature thylakoid membranes, this transition was identified as the ATPase denaturation band.

Visualization and characterization of prolamellar bodies with atomic force microscopy.

Prolamellar bodies (PLBs) isolated from etiolated wheat seedlings were studied with the use of atomic force microscopy (AFM), transmission electron microscopy (TEM) and fluorescence spectroscopy. With AFM, PLBs were seen as spherical structures about 1-2μm in diameter, more elastic than mica and poly-l-lysine substrate. TEM analyses confirmed that PLBs of wheat leaf etioplasts also had an average diameter of appr. 1μm. Illumination induced the photoreduction of photoactive protochlorophyllide (Pchlide), i.e. Pchlide bound to protochlorophyllide oxidoreductase, which was shown in fluorescence spectra. The photoreduction was followed by the disruption of PLB structures, which started with the enlargement of PLB spheres and then their fragmentation into small balls as seen with AFM. Light-induced vesicle formation and the outgrowth of lamellar (pro)thylakoid membranes on the PLB surface were also confirmed by TEM analyses, and resulted in the apparent enlargement of the PLB diameter. The blue-shift of the fluorescence emission maximum of chlorophyllide observed for PLBs at room temperature after Pchlide photoreduction was completed within 25min. However, structural changes in PLBs were still observed after the completion of the blue-shift. The incubation of PLBs in darkness with HgCl2 also resulted in PLB enlargement and a loosening of their structure. AFM provides a unique opportunity to observe PLBs at a physiological temperature without the necessity of fixation.

Role of Aquatic Macrophytes in Biogeochemical Cycling of Heavy Metals, Relevance to Soil-Sediment Continuum Detoxification and Ecosystem Health

The wetland sediments and soils of floodplains play an important role in the biogeocycling of heavy metals. Aquatic macrophytes are important components of wetland ecosystems. Here, the role of aquatic macrophytes in metal dynamics in wetland sediments is presented. The mechanisms of tolerance to metals in aquatic plants, including metal immobilization, chelation, translocation, and metabolic adaptations, are reviewed based on selected examples from the recent literature. The role of both photosynthetic activity and competitive/synergistic effects of the elements available to aquatic macrophytes in the circulation and deposition of metals are discussed in terms of the functioning of wetland ecosystems and phytoremediation.

MGDG, PG and SQDG regulate the activity of light-dependent protochlorophyllide oxidoreductase

Light-dependent protochlorophyllide oxidoreductase (POR) is a plant enzyme involved in the chlorophyll biosynthesis pathway. POR reduces one of the double bonds of the protochlorophyllide (Pchlide) using NADPH and light. In the present study, we found out that phosphatidylglycerol and sulfoquinovosyl diacylglycerol are allosteric regulators of the nucleotide binding, which increase the affinity towards NADPH a 100-fold. Moreover, we showed for the first time that NADH can, like NADPH, form active complexes with Pchlide and POR, however, at much higher concentrations. Additionally, monogalactosyldiacylglycerol (MGDG) was shown to be the main factor responsible for the red shift of the fluorescence emission maximum of Pchlide:POR:NADPH complexes. Importantly, the emission maximum at 654 nm was obtained only for the reaction mixtures supplemented with MGDG and at least one of the negatively charged plant lipids. Moreover, the site-directed mutagenesis allowed us to identify amino acid residues that may be responsible for lipid binding and Pchlide coordination. Our experiments allowed us to identify six different Pchlide:POR complexes that differ in the fluorescence emission maxima of the pigment. The results presented here reveal the contribution of thylakoid lipids in the regulation of the chlorophyll biosynthesis pathway; however, the molecular mechanisms of this process are to be clarified.

Protochlorophyllide in model systems--an approach to in vivo conditions.

Absorption and fluorescence properties of protochlorophyllide (Pchlide) monomers and aggregates in various model systems are presented in this study. The absorption and fluorescence maxima, and fluorescence lifetimes of Pchlide monomers were not dependent on liposome composition. Fluorescence quenching experiments using KI and SASLs as fluorescence quenchers, revealed that Pchlide molecules entered a lipid bilayer and were localized close to the polar lipid headgroup area. The process of Pchlide aggregation was evident for high (i.e. at least 9 mol%) Pchlide content in liposomes prepared from galactolipids. To our knowledge, this is the first study of Pchlide aggregation in membrane-mimicking model systems. The aggregates showed absorption maxima at 480 and 650 nm. Fluorescence of the aggregates measured for excitation at 480 nm had a maximum at 656 nm and was characterized with two fluorescence lifetime components, i.e. 0.1 and 1-2 ns. Pchlide aggregates observed in the buffer had similar position of absorption and fluorescence bands to those observed in liposomes, although the overall fluorescence intensity was considerably lower. Some differences in the relative intensity of Soret absorption bands were observed. These results showed that the presence of liposomes decreased the efficiency of the process of Pchlide aggregation. Water bound at the interface region of AOT/isooctane/water reversed micelles induced disaggregation of the Pchlide aggregates indicating that Pchlide aggregates are buried into hydrophilic core of micelles. The results are discussed with respect to the role of lipids in Pchlide aggregation found in plant etioplasts and their significance for light-induced Pchlide photoreduction.

Insight into the oligomeric structure of PORA from A. thaliana

Light-dependent protochlorophyllide oxidoreductase (POR, E.C. 1.3.1.33) is a plant enzyme that directly needs light to conduct a biochemical reaction. In the present paper we confirmed that POR forms large oligomers in solution before binding of substrates. We carried out the research using different techniques: cross-linking, native gel electrophoresis and FRET measurements. Mass spectrometry analysis of the cross-link products provided the first structural data about the organisation of the oligomer of POR. The results indicated that the catalytic motifs of the adjacent subunits become close to each other upon binding of substrates. Moreover, we identified two mutations of POR that disturbed its oligomerisation properties: Δ85-88 and Δ240-270. Additionally, a complete loss of the catalytic activity was observed for the following mutations: Δ189-194, Δ240-270, Δ318-331 and Δ392-393.