Correlated Changes in the Activity,Amount of Protein,and Abundance of Transcript of NADPH:Protochlorophyllide Oxidoreductase and Chlorophyll Accumulation during Greening of Cucumber Cotyledons

Changes in the activity and abundance of NADPH:protochlorophyllide oxidoreductase (NPR) and the abundance of mRNA encoding it were examined during the greening of 5-d-old etiolated cucumber cotyledons under continuous illumination. To measure NPR activity in the extracts from fully greened tissues, we have developed an improved method of assay. Upon exposure of etiolated cotyledons to light, NPR activity decreased rapidly within the first 2 h of exposure. Thereafter, enzymatic activity increased transiently, reaching a submaximum level at 12 h, and decreased slowly. The level of immunodetectable NPR protein followed the same pattern of changes during 96 h of greening as observed for NPR activity. The NPR mRNA in etiolated cotyledons disappeared quickly in the 1st h of irradiation. However, the level of mRNA increased thereafter to reach 3-fold or more of the dark level at 12 h and then decreased. The changes in the activity, protein level, and mRNA level after the first rapid decreases corresponded chronologically and nearly paralleled the increase in the rate of chlorophyll accumulation. These findings suggest that the greening of cucumber cotyledons is regulated basically by the level of NPR protein without activation or repression of enzymatic activity and that NPR mRNA increased by light maintains the level of enzyme protein necessary for greening.     

Preferential regeneration of the NADPH: protochlorophyllide oxidoreductase oligomer complexes in pea epicotyls after bleaching

The regeneration and stability of the NADPH:protochlorophyllide oxidoreductase (POR, EC 1.3.1.33) enzyme complexes were studied in bleached epicotyls of 9-day-old dark-germinated pea ( Pisum sativum L. cv. Zsuzsi) seedlings. Middle segments were illuminated with 1300 µmol m⁻² s⁻¹photon flux density (PFD) white light and subsequently incubated in total darkness for 4–24 h at 24°C. Almost the full amount of protochlorophyllide (Pchlide) was degraded after 60 min illumination. The preferential regeneration of the 655 nm emitting Pchlide form was observed after 4 h dark incubation; the accumulation of the short-wavelength Pchlide form—dominating in epicotyls of dark-grown seedling—required 18–24 h dark. The Pchlide content of bleached samples was around 2.5% of that of the etiolated samples; after 4 h of dark incubation this value increased to 4–7%. Polyacrylamide gel electrophoresis and western blot showed that the amount of the POR protein decreased to about 50% during bleaching; after 4 h regeneration it reached almost the same level as that of dark-grown samples. We concluded that much more POR protein compared with Pchlide pigment remained stable during bleaching and the non-destroyed POR units were able to form preferentially oligomers during the dark-regeneration which could collect de novo synthesized Pchlide into 655 nm emitting complexes. These data indicate the high stability of the POR protein in pea epicotyls and the importance of the molecular environment in stimulating the aggregation of POR units.     

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.     

Delayed chlorophyll accumulation and pigment photodestruction in the epicotyls of dark-grown pea (Pisum sativum)

A comparison was performed of the tetrapyrrole transformations that occur upon irradiation of epicotyl or leaves of dark-grown Pisum sativum L. (var. Zsuzsi, Hungary). High performance liquid chromatography analysis after continuous or flash-irradiation showed that the biosynthetic pathway from protochlorophyllide (Pchlide) to chlorophyll (Chl) a was markedly slowed down at the step of the reduction of geranylgeranyl(gg)-Chl to dihydrogeranylgeranyl (dhgg)-Chl in epicotyls, whereas phytyl-Chl was synthesized in leaves subjected to the same light treatments. Quantitative pigment analysis during continuous irradiations of different intensities also showed that significant Pchlide photodestruction occurred in epicotyls even under weak light. When both Pchlide and chlorophyllide and/or chlorophylls were present in epicotyls, Pchlide photodestruction was faster under 630-nm light than under 670-nm light, which indicates that this process is most efficiently promoted by Pchlide excitation. Pre-incubation of epicotyl segments with 10 m M ascorbate partly alleviated pigment photodestruction in white light. It is concluded that formation of photoactive Pchlide–Pchlide oxidoreductase complexes is important to prevent fast pigment photooxidation after Pchlide accumulation in the dark.     

Integration of nuclear-encoded proteins into pea thylakoids with different pigment contents

The in vitro membrane integration of the light-harvesting protein of photosystem II (LHCP), the Rieske FeS protein of the cytochrome (Cyt) blf-complex, and the NADPH:protochlorophyllide oxidoreductase (Pchlide reductase) into pea thylakoids with different pigment composition was studied. Pea plants (Pisum sativum L. cv. Kelvedon Wonder) with different contents of chlorophyll (Chl) and carotenoids were obtained by growing the seedlings in a greenhouse or in weak red light with or without the herbicide Norflurazon, an inhibitor of carotenoid biosynthesis. Chloroplasts from untreated and Norflurazon-treated plants grown in weak red light contained approximately 29 and 14% of Chl compared to chloroplasts from untreated plants grown in the greenhouse. The corresponding carotenoid contents were 66 and 5%. Following an integration reaction using LHCP precursor protein and chloroplast lysate, thylakoids from untreated and Norflurazon-treated plants grown in weak red light contained approximately 30 and 5% of protease-protected LHCP, respectively, compared to thylakoids of untreated plants grown in a greenhouse. In contrast to LHCP, the in vitro assembly of the Pchlide reductase was only sligthly reduced in chloroplast lysates of plants grown in weak red light compared to greenhouse-grown plants. In chloroplast lysates of Norflurazon-treated plants, however, the amount of membrane associated, protease-protected Pchlide reductase was reduced to 32% of the amount in untreated plants grown under the same light conditions. In contrast, the integration of the Rieske FeS protein occurred to almost similar levels irrespective of light conditions and herbicide treatments. Reconstitution assays where stroma from Norflurazon-treated plants was added to thylakoids from untreated plants, showed that the herbicide did not affect any stromal component(s) vital for the insertion reaction. Removal of samples during the integration reaction of LHCP showed that no degradation of the protein occurred during the assay. Neither was the assembled protein degraded up to 24 h after the termination of the assay. This indicates that growing plants in weak red light, with or without Norflurazon treatment, mainly affected the primary step in thylakoid assembly of LHCP, i.e. the insertion reaction into the membrane. The results further indicate that proteins normally bound to pigments also require pigments for membrane recognition or integration.     

Differential effects of benzyladenine and potassium on DNA, RNA, protein and chlorophyll contents and on expansion growth of detached cucumber cotyledons in the dark and light

Benzyladenine (BA) and KCl were applied to detached cucumber ( Cucumis sativus L. cv. Ohio) cotyledons in continuous light or in the dark with subsequent light. BA brought about an increase in fresh weight and in DNA, RNA and carotenoid contents in both treatments. KCl did not cause an increase in fresh weight and cellular constituents in the dark, but it did result in an increased fresh weight and DNA content after illumination or in continuous light. BA + KCl treatment resulted in increased carotenoid and DNA contents in the dark, and in increases in fresh weight and all cellular constituents upon subsequent exposure to light. The effects of BA and BA + KCl on growth and chlorophyll synthesis decreased with cotyledon age.
BA pretreatment in the dark eliminated the lag phase in chlorophyll synthesis and increased the rate of synthesis. Treatment in continuous light had little effect. KCl did not shorten the lag phase in chlorophyll synthesis, but it stimulated the rate of synthesis in the light. Dark pretreatment with BA + KCl markedly increased the effect of BA on chlorophyll synthesis. Chlorophyll content and fresh weight were higher in cotyledons treated with BA followed by KCl than in cotyledons treated in the reverse order. These results suggest that growth and greening in cucumber cotyledons are primarily controlled by BA and that KCl intensifies the BA effect after irradiation.     

Identification and characterization of the product release steps within the catalytic cycle of protochlorophyllide oxidoreductase

The chlorophyll biosynthetic enzyme protochlorophyllide reductase (POR) catalyzes the reduction of protochlorophyllide (Pchlide) into chlorophyllide (Chlide) with reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. POR is a light-driven enzyme, which has provided a unique opportunity to trap intermediates and identify different steps in the reaction pathway by initiating catalysis with illumination at low temperatures. In the present work we have used a thermophilic form of POR, which has an increased conformational rigidity at comparable temperatures, to dissect and study the final stages of the reaction where protein dynamics are proposed to play an important role in catalysis. Low-temperature fluorescence and absorbance measurements have been used to demonstrate that the reaction pathway for this enzyme consists of two additional "dark" steps, which have not been detected in previous studies. Product binding studies were used to show that spectroscopically distinct Chlide species could be observed and were dependent on whether the NADPH or NADP(+) cofactor was present. As a result we have been able to identify the intermediates that are observed during the latter stages of the POR catalytic cycle and have shown that they are formed via a series of ordered product release and cofactor binding events. These events involve release of NADP(+) from the enzyme and its replacement by NADPH, before release of the Chlide product has taken place. Following release of Chlide, the subsequent binding of Pchlide allows the next catalytic cycle to proceed.     

POR - import and membrane association of a key element in chloroplast development

The development of proplastids or etioplasts to chloroplast is visualized by the accumulation of chlorophyll in leaves of higher plants. The biosynthesis of chlorophyll includes a light-dependent reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide). This light-dependent step is catalysed by the nucleus-encoded NADPH:Pchlide oxidoreductase (POR, EC 1.6.99.1). POR is active within plastids and therefore has to be translocated over the plastid envelope membranes. The import of chloroplast proteins seems to follow a general import pathway using translocons at the outer and inner envelope membrane. POR cross-linking to Toc75, one of the major translocon components at the outer envelope membrane, indicates its use of the general import pathway. However, since variations exist within the so-called general import pathway one has to consider previous data suggesting a novel totally Pchlide-dependent import pathway of one POR isoform, PORA. The suggested Pchlide dependency of POR import is discussed since recent observations contradict this idea. In the stroma the POR transit peptide is cleaved off and the mature POR protein is targeted to the plastid inner membranes. The correct and stable association of POR to the membrane requires the cofactor NADPH. Functional activity of POR calls for formation of an NADPH–Pchlide–POR complex, a formation that probably takes place after the membrane association and is dependent on a phosphorylation reaction.     

Immunodetection and photostability of NADPH-protochlorophyllide oxidoreductase in Pinus pinea L.

Antibody against the light-dependent NADPH-protochlorophyllide oxidoreductase of oat was used to detect a protein of the same molecular weight in cotyledons of 40-day-old dark-grown seedlings of Pinus pinea L. Exposure of the seedlings to light resulted in a rapid decrease in protochlorophyllide content without the concomitant decrease in 38 kDa protein which is observed on transfer of dark-grown angiosperm seedlings to light. The stability of the light-dependent NADPH-protochlorophyllide oxidoreductase in pine in the absence of accumulated substrate is consistent with either (1) a different mechanism of regulation of chlorophyll synthesis in gymnosperms or (2) a higher proportion of stable extra-plastidic protein reacting with the antibody to the light-dependent NADPH-protochlorophyllide oxidoreductase than is the case in angiosperms.Abbreviations Chl chlorophyll - Chlide chlorophyllide - NADPH-Pchlide oxidoreductase NADPH protochlorophyllide oxidoreductase - NC nitrocellulose - PBS phosphate buffered saline - Pchlide protochlorophyllide - SDS sodum dodecyl sulphate - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Purification of NADPH-P450 reductase (NPR) from Xenopus laevis and the developmental change in NPR expression

NADPH-P450 reductase (NPR) was purified from hepatic microsomes of Xenopus laevis. The electron transfer activity of purified NPR was 23.8 units/min/mg with horse cytochrome c. The aminopyrine demethylation activity of rat CYP2B1 with Xenopus NPR was 58.1 nmol/min/nmol. The corresponding cDNA was isolated from Xenopus liver. The homology in amino acid sequence deduced from NPR cDNA isolated from Xenopus liver was 80%, 78%, and 81% with human, rat, and rabbit NPR, respectively. Antibody against Xenopus NPR was prepared. The expression of NPR was investigated in various tissues and in early development by Western blotting. NPR was most abundantly expressed in the kidney, followed by the liver, lung, and heart. The brain had very low levels of NPR. The level of NPR protein was almost the same at all stages, 2-cell stage (st. 2), blastula (st. 8), gastrula (st. 12), tail bud (st. 26) and larva (st.35), examined in this study. We further investigated the distribution of NPR using whole-mount in situ hybridization. NPR mRNA was expressed in cement gland, lens placode, ear vesicle, mesencephalon, rhombencephalon, lymphatic vessel, and heart anlage in the embryo at stage 29. Xenopus NPR has similar properties to mouse and rat NPRs. Localization of NPR in Xenopus embryo was consistent with the abnormal region caused by NPR deficiency in mice.     

Novel Insights into the Enzymology,Regulation and Physiological Functions of Light-dependent Protochlorophyllide Oxidoreductase in Angiosperms

The reduction of protochlorophyllide (Pchlide) is a key regulatory step in the biosynthesis of chlorophyll in phototrophic organisms. Two distinct enzymes catalyze this reduction; a light-dependent NADPH:protochlorophyllide oxidoreductase (POR) and light-independent Pchlide reductase (DPOR). Both enzymes are widely distributed among phototrophic organisms with the exception that only POR is found in angiosperms and only DPOR in anoxygenic photosynthetic bacteria. Consequently, angiosperms become etiolated in the absence of light, since the reduction of Pchlide in angiosperms is solely dependent on POR. In eukaryotic phototrophs, POR is a nuclear-encoded single polypeptide and post-translationally imported into plastids. POR possesses unique features, its light-dependent catalytic activity, accumulation in plastids of dark-grown angiosperms (etioplasts) via binding to its substrate, Pchlide, and cofactor, NADPH, resulting in the formation of prolamellar bodies (PLBs), and rapid degradation after catalysis under subsequent illumination. During the last decade, considerable progress has been made in the study of the gene organization, catalytic mechanism, membrane association, regulation of the gene expression, and physiological function of POR. In this review, we provide a brief overview of DPOR and then summarize the current state of knowledge on the biochemistry and molecular biology of POR mainly in angiosperms. The physiological and evolutional implications of POR are also discussed.     

Loss of fumarylacetoacetate hydrolase causes light‐dependent increases in protochlorophyllide and cell death in Arabidopsis

Fumarylacetoacetate hydrolase (FAH) catalyses the final step of the tyrosine degradation pathway, which is essential to animals but was of unknown importance in plants until we found that mutation of Short‐day Sensitive Cell Death1 (SSCD1), encoding Arabidopsis FAH, results in cell death under short‐day conditions. The sscd1 mutant accumulates succinylacetone (SUAC), an abnormal metabolite caused by loss of FAH. Succinylacetone is an inhibitor of δ‐aminolevulinic acid (ALA) dehydratase (ALAD), which is involved in chlorophyll (Chl) biosynthesis. In this study, we investigated whether sscd1 cell death is mediated by Chl biosynthesis and found that ALAD activity is repressed in sscd1 and that protochlorophyllide (Pchlide), an intermediate of Chl biosynthesis, accumulates at lower levels in etiolated sscd1 seedlings. However, it was interesting that Pchlide in sscd1 might increase after transfer from light to dark and that HEMA1 and CHLH are upregulated in the light–dark transition before Pchlide levels increased. Upon re‐illumination after Pchlide levels had increased, reactive oxygen species marker genes, including singlet oxygen‐induced genes, are upregulated, and the sscd1 cell death phenotype appears. In addition, Arabidopsis WT seedlings treated with SUAC mimic sscd1 in decline of ALAD activity and accumulation of Pchlide as well as cell death. These results demonstrate that increase in Pchlide causes cell death in sscd1 upon re‐illumination and suggest that a decline in the Pchlide pool due to inhibition of ALAD activity by SUAC impairs the repression of ALA synthesis from the light–dark transition by feedback control, resulting in activation of the Chl biosynthesis pathway and accumulation of Pchlide in the dark.     

Tissue-Specific Features of Pigment Biogenesis in Coleoptiles of Greening Etiolated Seedlings of Cereals

Biogenesis of the pigment apparatus was studied in coleoptiles of postetiolated barley seedlings (Hordeum vulgare L.) and triticale (Triticale), differing in chlorophyll content, during growing in a “ light-darkness” regime with a 16-h photoperiod. Photoactive protochlorophyllide with a fluorescence maximum at 655 nm (Pchlide655), which accumulates in coleoptiles of etiolated seedlings, was converted in the light into a chlorophyll pigment with a fluorescence maximum at 690 nm (excitation at 440 nm, temperature ?196°C). The spectral transition 690 nm → 675 nm forms was completed in darkness for 15 min illumination. There was almost no resynthesis of new portions of Pchlide655 in coleoptiles under darkness conditions, even after a 5–6-h darkness period after brief illumination of seedlings with flashes of white light. Chlorophyllide (Chlide) formed from Pchlide655 was not esterified and was destroyed both in the light (4 h, 1.0–1.5 klx) and darkness. In coleoptiles of greening etiolated seedlings, chlorophyll formation started only by 24 h of illumination. The instability of the chlorophyll pigment formed after etiolation indicates that plastids of coleoptiles do not contain the system of chlorophyll biosynthesis centers typical of leaves, which are bound to membranes and protect pigment from destruction.     

Differential operation of dual protochlorophyllide reductases for chlorophyll biosynthesis in response to environmental oxygen levels in the cyanobacterium Leptolyngbya boryana

Most oxygenic phototrophs, including cyanobacteria, have two structurally unrelated protochlorophyllide (Pchlide) reductases in the penultimate step of chlorophyll biosynthesis. One is light-dependent Pchlide reductase (LPOR) and the other is dark-operative Pchlide reductase (DPOR), a nitrogenase-like enzyme assumed to be sensitive to oxygen. Very few studies have been conducted on how oxygen-sensitive DPOR operates in oxygenic phototrophic cells. Here, we report that anaerobic conditions are required for DPOR to compensate for the loss of LPOR in cyanobacterial cells. An LPOR-lacking mutant of the cyanobacterium Leptolyngbya boryana (formerly Plectonema boryanum) failed to grow in high light conditions and this phenotype was overcome by cultivating it under anaerobic conditions (2% CO(2)/N(2)). The critical oxygen level enabling the mutant to grow in high light was determined to be 3% (v/v). Oxygen-sensitive Pchlide reduction activity was successfully detected as DPOR activity in cell-free extracts of anaerobically grown mutants, whereas activity was undetectable in the wild type. The content of two DPOR subunits, ChlL and ChlN, was significantly increased in mutant cells compared with wild type. This suggests that the increase in subunits stimulates the DPOR activity that is protected efficiently from oxygen by anaerobic environments, resulting in complementation of the loss of LPOR. These results provide important concepts for understanding how dual Pchlide reductases operate differentially in oxygenic photosynthetic cells grown under natural environments where oxygen levels undergo dynamic changes. The evolutionary implications of the coexistence of two Pchlide reductases are discussed.     

Abstracts of papers read at the winter meeting at the university of Newcastle Upon Tyne

Young plants of Laminaria hyperborea collected from the field were grown for 2·5–4 weeks in blue, green, red and white (simulated underwater) light fields at 5, 20 and 100 μmol m^-2s^-1. The absolute concentrations of all pigments showed little variation with irradiance in green and white light, but decreased in high irradiances of red and blue light. The ratio of fucoxanthin to chlorophyll a also increased in the latter treatments, as did the chlorophyll c:a ratio in bright red light. There was little difference in the action spectrum for photosynthesis between the different light qualities at any one irradiance, but the action spectra for plants grown at 100 μmol m^-2s^-1 showed deeper troughs and higher peaks than those for plants grown at lower irradiances. Gross photosynthesis per unit of thallus area at 10 μmol m^-2s^-1 decreased in plants with low total pigment concentrations, but the photosynthesis per unit of pigment concentration increased. This suggestion of self-shading of pigment molecules within the algal thalli was supported by a flattening of the action spectrum in plants with higher chlorophyll a contents. The variations observed between the action spectra for different plants could thus be attributed to the decrease in pigment content at high irradiances, and not to the light quality in which the plants were grown.     

Effects of growth regulators and light on chloroplasts NAD(P)H dehydrogenase activities of senescent barley leaves

The activities NADH and NADPH dehydrogenases were measured with ferricyanide as electron-acceptor (NADH-FeCN-ox and NADPH-FeCN-ox, respectively) in mitochondria-free chloroplasts of barley leaf segments after receiving various treatments affecting senescence. NADPH-FeCN-ox declined during senescence in the dark, in a way similar to chlorophyll and Hill reaction, and increased when leaf segments were incubated at light. These results suggest that NADPH-FeCN-ox is related to some photosynthetic electron transporter activity (probably ferredoxin-NADP⁺ oxidoreductase). In contrast, NADH-FeCN-ox is notably stable during senescence in the dark and at light. This activity increased during incubation with kinetin or methyl-jasmonate (Me-JA) but decreased when leaf segments were treated with abscisic acid (ABA). The effects of the inhibitors of protein synthesis cycloheximide and chloramphenicol suggest that the changes of NAD(P)H dehydrogenase activities may depend on protein synthesis in chloroplasts. In senescent leaf, chloroplast NADH dehydrogenase might be a way to dissipate NADH produced in the degradation of excess carbon which is released from the degradation of amino acids.Abbreviations ABA abscisic acid - DCPIP 2,6-dichlorophenol-indo-phenol - DOC deoxycholate - Me-JA methyl jasmonate - NADH-FeCN-ox NADH ferricyanide oxidoreductase - NADPH-FeCN-ox NADPH ferricyanide oxidoreductase     

In situ conversion of protochlorophyllide b to protochlorophyllide a in barley. Evidence for a novel role of 7-formyl reductase in the prolamellar body of etioplasts

We recently put forth a model of a protochlorophyllide (Pchlide) light-harvesting complex operative during angiosperm seedling de-etiolation (Reinbothe, C., Lebedev, N., and Reinbothe, S. (1999) Nature 397, 80-84). This model, which was based on in vitro reconstitution experiments with zinc analogs of Pchlide a and Pchlide b and the two NADPH:protochlorophyllide oxidoreductases (PORs), PORA and PORB, of barley, predicted a 5-fold excess of Pchlide b, relative to Pchlide a, in the prolamellar body of etioplasts. Recent work (Scheumann, V., Klement, H., Helfrich, M., Oster, U., Schoch, S., and Rüdiger, W. (1999) FEBS Lett. 445, 445-448), however, contradicted this model and reported that Pchlide b would not be present in etiolated plants. Here we demonstrate that Pchlide b is an abundant pigment in barley etioplasts but is rather metabolically unstable. It is rapidly converted to Pchlide a by virtue of 7-formyl reductase activity, an enzyme that had previously been implicated in the chlorophyll (Chl) b to Chl a reaction cycle. Our findings suggest that etiolated plants make use of 7-formyl reductase to fine tune the levels of Pchlide b and Pchlide a and thereby may regulate the steady-state level of light-harvesting POR-Pchlide complex.     

Kinetic characterisation of the light-driven protochlorophyllide oxidoreductase (POR) from Thermosynechococcus elongatus.

The light-driven enzyme NADPH:protochlorophyllide oxidoreductase (POR) catalyses the reduction of the C17-C18 double bond of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), which is a key regulatory step in the chlorophyll biosynthesis pathway. POR from the thermophilic cyanobacterium Thermosynechococcus elongatus is an attractive system for following the reaction and in the present work we have carried out a detailed steady state kinetic characterisation of this enzyme. The thermophilic POR was shown to have maximal activity at approximately 50 degrees C, which is similar to the growth temperature of the organism. The V(max) was calculated to be 0.53 microM min(-1) and the K(m) values for NADPH and Pchlide were 0.013 microM and 1.8 microM, respectively. The binding properties for both substrates as well as the NADP(+) product have been analysed by using fluorescence emission measurements, which have allowed the dissociation constants for binding to be calculated. These results represent the first steady state kinetic characterisation of a thermophilic version of POR.     

Nuclear localization of NPR1 is required for activation of PR gene expression

Systemic acquired resistance (SAR) is a broad-spectrum resistance in plants that involves the upregulation of a battery of pathogenesis-related (PR) genes. NPR1 is a key regulator in the signal transduction pathway that leads to SAR. Mutations in NPR1 result in a failure to induce PR genes in systemic tissues and a heightened susceptibility to pathogen infection, whereas overexpression of the NPR1 protein leads to increased induction of the PR genes and enhanced disease resistance. We analyzed the subcellular localization of NPR1 to gain insight into the mechanism by which this protein regulates SAR. An NPR1-green fluorescent protein fusion protein, which functions the same as the endogenous NPR1 protein, was shown to accumulate in the nucleus in response to activators of SAR. To control the nuclear transport of NPR1, we made a fusion of NPR1 with the glucocorticoid receptor hormone binding domain. Using this steroid-inducible system, we clearly demonstrate that nuclear localization of NPR1 is essential for its activity in inducing PR genes.