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.     

Prolonged salt stress alters the ratios of protochlorophyllide spectral forms in dark-grown wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) and influences chlorophyll <Emphasis Type="Italic">a</Emphasis> accumulation following irradiation

To examine the effects of salt stress on dark-grown wheat (Triticum aestivum), seedlings of the salt-tolerant cultivar Sids 1 and the susceptible cultivar Giza 168 were grown in darkness for 14 days in nutrient solution with and without 200 mM of supplementary salt (100 mM of NaCl and 100 mM of KCl). During this time, we monitored their protochlorophyllide (Pchlide) contents, ratios of photoactive to non-photoactive forms of Pchlide (from 655/633-nm emission ratios in their 77 K fluorescence emission spectra) and (following flash irradiation) ratios of newly formed chlorophyllide (Chlide) to non-photoactive Pchlide. In addition, the accumulation of chlorophyll a in leaf sections was monitored during prolonged (24 h) irradiation. The results depended on the developmental state of the seedlings. However, the salt stress treatment caused marked increases in both Pchlide contents in dark-grown leaves and in Chlide contents following irradiation of leaf sections of both cultivars. The ratio of phototransformable to non-phototransformable Pchlide and the abundance of newly formed Chlide were also increased by the salt stress. Further, leaves of salt-stressed seedlings consistently accumulated more chlorophyll a than leaves of unstressed seedlings when floating on the nutrient solution (with or without supplementary salt) in continuous white light. The findings are consistent with the hypothesis that increased levels of the long-wavelength form of Pchlide contribute to protective mechanisms against salt stress.     

Chlorophyll Formation in the YG-6 Mutant of Chlorella regularis: Spectral Characterization of Protochlorophyllide Phototransformation

Dark-grown YG-6 mutant cells of Chlorella regularis accumulateat least two forms of phototransformable protochlorophyllide(Pchlide) with in vivo absorption maxima at 634 nm (Pchlide634) and 650 nm (Pchlide 650). Difference spectrophotometricanalyses and the action spectra showed that Pchlide 634 is firsttransformed into the 648 nm form and then phototransformed intochlorophyllide (Chlide) 672 nm. Pchlide 650 is phototransformedinto Chlide 685 which then shifts towards short wavelength-formingChlide 667 in the subsequent dark stage (Shibata shift). Pchlide650 is regenerated at the expense of photoinactive Pchlide 632.In washed cells after the phototransformation, the Shibata shiftwas accelerated. Freezing/thawing treatment in the dark causedconversion of phototransformable Pchlide 650 into photoinactivePchlide 633, but phototransformation activity of Pchlide 634still partly remained. These results suggest that in the final step of light-dependentchlorophyll formation in the YG-6 mutant of C. regularis, twosequentially and functionally separate routes are present: (1) Pchlide 634 Pchlide 648 Chlide 672 Chlorophyll a. (2) Pchlide 650 Chlide 685 Chlide 667 Chlorophyll a. (Received June 4, 1983; Accepted November 11, 1983)     

The stimulation of indoleacetic acid synthesis in bush bean plants (Phaseolus vulgaris L.) by naphthenates

A 12 hr seed soak in a solution of 100 ppm of potassium naphthenatesresulted in 140.5% stimulation of IAA synthesis determined in5–8 cm tips of epicotyls of 14-day-old dark-grown Phaseolusvulgaris seedlings. (Received September 1, 1973; )     

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.     

The cytokinin 2-isopentenyladenine causes partial reversion to skotomorphogenesis and induces formation of prolamellar bodies and protochlorophyllide657 in the lip1 mutant of pea

When grown in darkness the photomorphogenic lip 1 mutant of pea ( Pisum sativum L.) has a slender stem, expanded leaves, prolamellar body (PLB) lacking plastids with the size of chloroplasts and a low level of phytochrome A. The lack of PLBs in a dark-grown material ( lip 1) created a possibility to further study the regulation of their formation in relation to plant development. Inclusion of a cytokinin, 2-isopentenyladenine (2iP), in a medium supporting growth of the pea seedlings in darkness was found to reduce epicotyl length in the wild type. In lip 1 the formation of a slender stem was inhibited and a short epicotyl developed. Furthermore, leaf expansion was inhibited, the plastid size reduced and the formation of PLBs induced. The PLB formation in lip 1 was not accompanied by an increase in the amount of protochlorophyllide (Pchlide) or Pchilde oxidoreductase (POR). In the presence of 2iP the level of phytochrome A protein was increased in lip 1 and the POR mRNA levels decreased in both lip 1 and wild-type plants. The chloroplast characteristic trans -3-hexadecenoate acyl group of phosphatidylglycerol, present in the plastids of dark-grown lip 1, was not influenced by 2iP. Thus, not all photomorphogenic processes reacted similarly in the lip 1 mutant, but leaf expansion and plastid differentiation, including PLB formation, seemed to be regulated by the same signal transduction chain. Exogenously applied brassinolide could rescue neither dark- nor light-grown defects of the lip 1 mutant. Thus, cytokinins but not brassinolides seem to be involved in the regulation of certain characteristic traits of skotomorphogenesis in pea, including plastid development and PLB formation.     

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.     

Two NADPH:Protochlorophyllide Oxidoreductases in Barley: Evidence for the Selective Disappearance of PORA during the Light-Induced Greening of Etiolated Seedlings

Chlorophyll synthesis in barley is controlled by two different light-dependent NADPH:protochlorophyllide oxidoreductases, termed PORA and PORB. PORA is present abundantly in etioplasts but selectively disappears soon after the beginning of illumination. This negative light effect is mediated simultaneously at three different levels. First, the concentration of porA mRNA declines drastically during illumination of dark-grown seedlings. Second, the plastids' ability to import the precursor of PORA (pPORA) is reduced during the transition from etioplasts to chloroplasts. This effect is due to a rapid decline in the plastidic level of protochlorophyllide (Pchlide), which is required for the translocation of the pPORA. Third, PORA becomes selectively destabilized in illuminated seedlings. When illuminated, PORA-Pchlide-NADPH complexes formed in the dark photoreduce their Pchlide to Chlide and become simultaneously susceptible to attack by plastid proteases. The PORA-degrading protease activity is not detectable in etioplasts but is induced during illumination. In contrast to PORA, the second Pchlide-reducing enzyme, PORB, remains operative in both illuminated and green plants. Its translocation into plastids does not depend on its substrate, Pchlide.     

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.     

Protochlorophyllide and POR development in dark‐grown plants with different proportions of short‐wavelength and long‐wavelength protochlorophyllide spectral forms†

The effect of leaf developmental age on the protochlorophyllide (Pchlide) spectral forms and the expression of messenger RNA (mRNA) encoding NADPH‐protochlorophyllide oxidoreductase (POR) were investigated. Four plant species, maize, wheat, pea and the lip1 mutant of pea, known to have different composition of the spectral forms of Pchlide, were used. In very young plants short‐wavelength Pchlide with a fluorescence emission at 631 nm was dominating. Long‐wavelength Pchlide fluorescing mainly around 655 nm increased during development, which led to a relative decrease of the short‐wavelength forms. During ageing of the leaves, the short‐wavelength forms slightly increased again. The different proportions of short‐ and long‐wavelength Pchlide spectral forms were, however, found to vary with the developmental stage in a species specific pattern. The steady‐state level of POR mRNA and the amount of the POR protein were similar in species dominated by short‐wavelength forms and in species dominated with long‐wavelength forms. Even if POR is necessary for the formation of the long‐wavelength Pchlide form it is not the only limiting factor for formation of long‐wavelength Pchlide forms in mature plants.     

Root Graviresponsiveness and Cellular Differentiation in Wild-type and a Starchless Mutant of Arabidopsis thaliana

Primary roots of a starchless mutant of Arabidopsis thalianaL. are strongly graviresponsive despite lacking amyloplastsin their columella cells. The ultrastructures of calyptrogenand peripheral cells in wild-type as compared to mutant seedlingsare not significantly different. The largest difference in cellulardifferentiation in caps of mutant and wild-type roots is therelative volume of plastids in columella cells. Plastids occupy12.3% of the volume of columella cells in wild-type seedlings,but only 3.69% of columella cells in mutant seedlings. Theseresults indicate that: (1) amyloplasts and starch are not necessaryfor root graviresponsiveness; (2) the increase in relative volumeof plastids that usually accompanies differentiation of columellacells is not necessary for root graviresponsiveness; and (3)the absence of starch and amyloplasts does not affect the structureof calyptrogen (i.e. meristematic) and secretory (i.e. peripheral)cells in root caps. These results are discussed relative toproposed models for root gravitropism. Arabidopsis thaliana, gravitropism (root), plastids, root cap, stereology, ultrastructure     

Mechanistic Reappraisal of Early Stage Photochemistry in the Light-Driven Enzyme Protochlorophyllide Oxidoreductase

The light-driven enzyme protochlorophyllide oxidoreductase (POR) catalyzes the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide). This reaction is a key step in the biosynthesis of chlorophyll. Ultrafast photochemical processes within the Pchlide molecule are required for catalysis and previous studies have suggested that a short-lived excited-state species, known as I675*, is the first catalytic intermediate in the reaction and is essential for capturing excitation energy to drive subsequent hydride and proton transfers. The chemical nature of the I675* excited state species and its role in catalysis are not known. Here, we report time-resolved pump-probe spectroscopy measurements to study the involvement of the I675* intermediate in POR photochemistry. We show that I675* is not unique to the POR-catalyzed photoreduction of Pchlide as it is also formed in the absence of the POR enzyme. The I675* species is only produced in samples that contain both Pchlide substrate and Chlide product and its formation is dependent on the pump excitation wavelength. The rate of formation and the quantum yield is maximized in 50∶50 mixtures of the two pigments (Pchlide and Chlide) and is caused by direct energy transfer between Pchlide and neighboring Chlide molecules, which is inhibited in the polar solvent methanol. Consequently, we have re-evaluated the mechanism for early stage photochemistry in the light-driven reduction of Pchlide and propose that I675* represents an excited state species formed in Pchlide-Chlide dimers, possibly an excimer. Contrary to previous reports, we conclude that this excited state species has no direct mechanistic relevance to the POR-catalyzed reduction of Pchlide.     

POR hits the road: import and assembly of a plastid protein

The biosynthesis of chlorophyll is a strictly light-dependent multistep process in higher plants. The light-dependent step is catalysed by NADPH:protochlorophyllide oxidoreductase (POR, EC.1.6.99.1), which reduces protochlorophyllide (Pchlide) to chlorophyllide (Chlide). POR is nucleus-encoded and post-translationally imported into plastids. It has been proposed that the import of a POR protein isozyme (PORA) is totally dependent on Pchlide and uses a novel import pathway. This proposal is based on findings that PORA import only occurs in the presence of Pchlide and that the presence of overexpressed precursor of Rubisco small subunit (pSS), a protein which is known to use the general import pathway, does not outcompete PORA import. Another study demonstrated that POR precursor protein (pPOR) can be cross-linked to one of the components in the translocation machinery, Toc75, in the absence of Pchlide, and that its import can be outcompeted by the addition of the pSS. This indicates that pSS and pPOR may use the same translocation mechanism. Thus, POR does not necessarily need Pchlide for import – which is in contrast to earlier observations – and the exact POR import mechanism remains unresolved. Once in the stroma, the POR transit peptide is cleaved off and the mature POR protein is associated to the plastid inner membranes. Formation of the correct membrane–associated, thermolysin-protected assembly is strictly dependent of NADPH. As a final step, the formation of the NADPH-Pchlide-POR complex occurs. When POR accumulates in the membranes of proplastids, an attraction of monogalactosyl diacylglycerol (MGDG) can occur, leading to the formation of prolamellar bodies (PLBs) and the development of etioplasts in darkness.     

Root Graviresponsiveness and Columella Cell Structure in Carotenoid-deficient Seedlings of Zea mays

Root graviresponsiveness in normal and carotenoid-deficientmutant seedlings of Zea mays was not significantly different.Columella cells in roots of mutant seedlings were characterizedby fewer, smaller, and a reduced relative volume of plastidsas compared to columella cells of normal seedlings. Plastidsin columella cells of mutant seedlings possessed reduced amountsof starch. Although approximately 10 per cent of the columellacells in mutant seedlings lacked starch, their plastids werelocated at the bottom of the cell. These results suggest that(i) carotenoids are not necessary for root gravitropism, (ii)graviresponsiveness is not necessarily proportional to the size,number, or relative volume of plastids in columella cells, and(iii) sedimentation of plastids in columella cells may not resultdirectly from their increased density due to starch content.Plastids in columella cells of normal and mutant seedlings wereassociated with bands of microtubule-like structures, suggestingthat these structures may be involved in ‘positioning’plastids in the cell. Zea mays, graviperception, graviresponsiveness, carotenoids, vp-9 mutant, columella cell, roots     

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     

Overexpression of light-dependent PORA or PORB in plants depleted of endogenous POR by far-red light enhances seedling survival in white light and protects against photooxidative damage

The structurally related light-dependent protochlorophyllide (Pchlide) oxidoreductases PORA and PORB mediate the only light-requiring step in chlorophyll (Chl) biosynthesis in higher plants. Correlative evidence suggests that some in vivo functions of PORA and PORB may be unique, including a postulated photoprotective role for PORA. For example, wild-type Arabidopsis thaliana seedlings grown in non-photooxidative far-red light (cFR) resemble those grown in white light (WL), but they are yellow and do not green normally thereafter in WL. This defect is accompanied by the absence of detectable PORA and reduced levels of PORB expression. Here, direct evidence is provided that the presence of POR, either as PORA or PORB, can confer photoprotection in plants. In contrast to the wild-type, the plastids of transgenic PORA- or PORB-overexpressing Arabidopsis seedlings grown in cFR possess extensive prolamellar bodies. Upon a subsequent shift to WL, POR-overexpressing seedlings develop thylakoid membranes, accumulate large amounts of Chl and are viable at fluence rates lethal to the wild-type. Intriguingly, the plastid membrane architectures of greening transgenic seedlings seem to depend on whether PORA or PORB has been overproduced. POR-overexpressing seedlings shifted from cFR to WL of fluence rates from 20 to 500 μE m^–2 sec^–1 accumulate substantially higher amounts of Chl than does the wild-type. Furthermore, the WL fluence rate that permits maximal Chl accumulation increases from 8 μE m^–2 sec^–1 in the wild-type to 125 μE m^–2 sec^–1 in transgenic seedlings. POR overexpression during growth in cFR also correlates with a fourfold decrease in the steady-state content of Pchlide, a potentially lethal photosensitizer.     

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.