Protochlorophyllide synthesis by recombinant cyclases from eukaryotic oxygenic phototrophs and the dependence on Ycf54.

The unique isocyclic E ring of chlorophylls contributes to their role as light-absorbing pigments in photosynthesis. The formation of the E ring is catalyzed by the Mg-protoporphyrin IX monomethyl ester cyclase, and the O2-dependent cyclase in prokaryotes consists of a diiron protein AcsF, augmented in cyanobacteria by an auxiliary subunit Ycf54. Here, we establish the composition of plant and algal cyclases, by demonstrating the in vivo heterologous activity of O2-dependent cyclases from the green alga Chlamydomonas reinhardtii and the model plant Arabidopsis thaliana in the anoxygenic photosynthetic bacterium Rubrivivax gelatinosus and in the non-photosynthetic bacterium Escherichia coli. In each case, an AcsF homolog is the core catalytic subunit, but there is an absolute requirement for an algal/plant counterpart of Ycf54, so the necessity for an auxiliary subunit is ubiquitous among oxygenic phototrophs. A C-terminal ∼40 aa extension, which is present specifically in green algal and plant Ycf54 proteins, may play an important role in the normal function of the protein as a cyclase subunit.

Formation of prolamellar-body-like ultrastructures in etiolated cyanobacterial cells overexpressing light-dependent protochlorophyllide oxidoreductase in Leptolyngbya boryana.

Protochlorophyllide (Pchlide) reduction is the penultimate step of chlorophyll (Chl) biosynthesis, and is catalyzed by two evolutionarily unrelated enzymes: dark-operative Pchlide oxidoreductase (DPOR) and light-dependent Pchlide oxidoreductase (LPOR). Because LPOR is the sole Pchlide reductase in angiosperms, dark-grown seedlings of angiosperms become etiolated. LPOR exists as a ternary complex of Pchlide-NADPH-LPOR to form paracrystalline prolamellar bodies (PLBs) in etioplasts. Because LPOR is distributed ubiquitously across oxygenic phototrophs including cyanobacteria, it would be important to determine whether cyanobacterial LPOR has the ability to form PLBs. We isolated a DPOR-less transformant ΔchlL/LPORox, carrying a plasmid to overexpress cyanobacterial LPOR in the cyanobacterium Leptolyngbya boryana. The transformant did not produce Chl in the dark and became etiolated with an accumulation of Pchlide and LPOR. Novel PLB-like ultrastructures were observed in etiolated cells, which disappeared during the early stage of the light-dependent greening process. However, the rate of Chl production in the greening process of ΔchlL/LPORox was almost the same as that observed in the control cells, which carried an empty vector. An in vitro LPOR assay of extracts of dark-grown ΔchlL/LPORox cells suggested that the PLB-like structures are deficient in NADPH. Low-temperature fluorescence emission spectra of membrane fractions of the etiolated cells indicated the absence of the photoactive form of Pchlide, which was consistent with the inefficiency of the greening process. Cyanobacterial LPOR exhibited an intrinsic ability to form PLB-like ultrastructures in the presence of the co-accumulation of Pchlide; however, the PLB-like structure differed from the authentic PLB regarding NADPH deficiency.

Processing bodies control the selective translation for optimal development of Arabidopsis young seedlings.

Germinated plant seeds buried in soil undergo skotomorphogenic development before emergence to reach the light environment. Young seedlings transitioning from dark to light undergo photomorphogenic development. During photomorphogenesis, light alters the transcriptome and enhances the translation of thousands of mRNAs during the dark-to-light transition in Arabidopsis young seedlings. About 1,500 of these mRNAs have comparable abundance before and after light treatment, which implies widespread translational repression in dark-grown seedlings. Processing bodies (p-bodies), the cytoplasmic granules found in diverse organisms, can balance the storage, degradation, and translation of mRNAs. However, the function of p-bodies in translation control remains largely unknown in plants. Here we found that an Arabidopsis mutant defective in p-body formation (Decapping 5; dcp5-1) showed reduced fitness under both dark and light conditions. Comparative transcriptome and translatome analyses of wild-type and dcp5-1 seedlings revealed that p-bodies can attenuate the premature translation of specific mRNAs in the dark, including those encoding enzymes for protochlorophyllide synthesis and PIN-LIKES3 for auxin-dependent apical hook opening. When the seedlings protrude from soil, light perception by photoreceptors triggers a reduced accumulation of p-bodies to release the translationally stalled mRNAs for active translation of mRNAs encoding proteins needed for photomorphogenesis. Our data support a key role for p-bodies in translation repression, an essential mechanism for proper skotomorphogenesis and timely photomorphogenesis in seedlings.

Monogalactosyldiacylglycerol Facilitates Synthesis of Photoactive Protochlorophyllide in Etioplasts.

Cotyledon cells of dark-germinated angiosperms develop etioplasts that are plastids containing unique internal membranes called prolamellar bodies (PLBs). Protochlorophyllide (Pchlide), a precursor of chlorophyll, accumulates in PLBs and forms a ternary complex with NADPH and light-dependent NADPH:protochlorophyllide oxidoreductase (LPOR), which allows for the rapid formation of chlorophyll after illumination while avoiding photodamage. PLBs are 3D lattice structures formed by the lipid bilayer rich in monogalactosyldiacylglycerol (MGDG). Although MGDG was found to be required for the formation and function of the thylakoid membrane in chloroplasts in various plants, the roles of MGDG in PLB formation and etioplast development are largely unknown. To analyze the roles of MGDG in etioplast development, we suppressed MGD1 encoding the major isoform of MGDG synthase by using a dexamethasone-inducible artificial microRNA in etiolated Arabidopsis (Arabidopsis thaliana) seedlings. Strong MGD1 suppression caused a 36% loss of MGDG in etiolated seedlings, together with a 41% decrease in total Pchlide content. The loss of MGDG perturbed etioplast membrane structures and impaired the formation of the photoactive Pchlide-LPOR-NADPH complex and its oligomerization, without affecting LPOR accumulation. The MGD1 suppression also impaired the formation of Pchlide from protoporphyrin IX via multiple enzymatic reactions in etioplast membranes, which suggests that MGDG is required for the membrane-associated processes in the Pchlide biosynthesis pathway. Suppressing MGD1 at several germination stages revealed that MGDG biosynthesis at an early germination stage is particularly important for Pchlide accumulation. MGDG biosynthesis may provide a lipid matrix for Pchlide biosynthesis and the formation of Pchlide-LPOR complexes as an initial step of etioplast development.

Conserved residues in Ycf54 are required for protochlorophyllide formation in Synechocystis sp. PCC 6803.

Chlorophylls (Chls) are modified tetrapyrrole molecules, essential for photosynthesis. These pigments possess an isocyclic E ring formed by the Mg-protoporphyrin IX monomethylester cyclase (MgPME-cyclase). We assessed the in vivo effects of altering seven highly conserved residues within Ycf54, which is required for MgPME-cyclase activity in the cyanobacterium SynechocystisSynechocystis strains harbouring the Ycf54 alterations D39A, F40A and R82A were blocked to varying degrees at the MgPME-cyclase step, whereas the A9G mutation reduced Ycf54 levels by ∼75%. Wild-type (WT) levels of the cyclase subunit CycI are present in strains with D39A and F40A, but these strains have lowered cellular Chl and photosystem accumulation. CycI is reduced by ∼50% in A9G and R82A, but A9G has no perturbations in Chl or photosystem accumulation, whilst R82A contains very little Chl and few photosystems. When FLAG tagged and used as bait in pulldown experiments, the three mutants D39A, F40A and R82A were unable to interact with the MgPME-cyclase component CycI, whereas A9G pulled down a similar level of CycI as WT Ycf54. These observations suggest that a stable interaction between CycI and Ycf54 is required for unimpeded Pchlide biosynthesis. Crystal structures of the WT, A9G and R82A Ycf54 proteins were solved and analysed to investigate the structural effects of these mutations. A loss of the local hydrogen bonding network and a reversal in the surface charge surrounding residue R82 are probably responsible for the functional differences observed in the R82A mutation. We conclude that the Ycf54 protein must form a stable interaction with CycI to promote optimal Pchlide biosynthesis.

Salt-stress induced modulation of chlorophyll biosynthesis during de-etiolation of rice seedlings.

Chlorophyll biosynthesis in plants is subjected to modulation by various environmental factors. To understand the modulation of the chlorophyll (Chl) biosynthesis during greening process by salt, 100-200 mM NaCl was applied to the roots of etiolated rice seedlings 12 h prior to the transfer to light. Application of 200 mM NaCl to rice seedlings that were grown in light for further 72 h resulted in reduced dry matter production (-58%) and Chl accumulation (-66%). Ionic imbalance due to salinity stress resulted in additional downregulation (41-45%) of seedling dry weight, Chl and carotenoid contents over and above that of similar osmotic stress induced by polyethylene glycol. Downregulation of Chl biosynthesis may be attributed to decreased activities of Chl biosynthetic pathway enzymes, i.e. 5-aminolevulinic acid (ALA) dehydratase (EC-2.4.1.24), porphobilinogen deaminase (EC-4.3.1.8), coproporphyrinogen III oxidase (EC-1.3.3.3), protoporphyrinogen IX oxidase (EC-1.3.3.4), Mg-protoporphyrin IX chelatase (EC-6.6.1.1) and protochlorophyllide oxidoreductase (EC-1.3.33.1). Reduced enzymatic activities were due to downregulation of their protein abundance and/or gene expression in salt-stressed seedlings. The extent of downregulation of ALA biosynthesis nearly matched with that of protochlorophyllide and Chl to prevent the accumulation of highly photosensitive photodynamic tetrapyrroles that generates singlet oxygen under stress conditions. Although, ALA synthesis decreased, the gene/protein expression of glutamyl-tRNA reductase (EC-1.2.1.70) increased suggesting it may play a role in acclimation to salt stress. The similar downregulation of both early and late Chl biosynthesis intermediates in salt-stressed seedlings suggests a regulatory network of genes involved in tetrapyrrole biosynthesis.

Light piping activates chlorophyll biosynthesis in the under-soil hypocotyl section of bean seedlings.

Protochlorophyllide (Pchlide), protochlorophyll (Pchl) and chlorophyll (Chl) contents, their distribution and native arrangements were studied in under-soil hypocotyl segments of 4-, 7- and 14-day-old bean (Phaseolus vulgaris L. cv. Magnum) seedlings. The plants were grown in general potting soil under natural illumination conditions in pots. For sample collection, the pots were transferred into dark-room where all manipulations were done under dim green light. The pigments were extracted with acetone; phase separation was used to identify the Pchl contents. Fluorescence microscopic studies were done and 77K fluorescence emission spectra were recorded. Using a special setup of a spectrofluorometer, the vertical light piping properties of the above-soil shoots were measured. The segments in the 5-7 cm deep soil region contained Pchlide and Pchl in 4- and 7-day-old seedlings and the segments towards the soil surface contained Chl in increasing amounts. In parallel with the pith degradation of hypocotyls, the Chl content of elder seedlings increased in the deeper under-soil segments. These results prove that the tissue structure of the shoot ensures light piping thus greening process and chloroplast formation can take place even in under-soil organs not directly exposed to light.

Ethylene-orchestrated circuitry coordinates a seedling's response to soil cover and etiolated growth.

The early life of terrestrial seed plants often starts under the soil in subterranean darkness. Over time and through adaptation, plants have evolved an elaborate etiolation process that enables seedlings to emerge from soil and acquire autotrophic ability. This process, however, requires seedlings to be able to sense the soil condition and relay this information accordingly to modulate both the seedlings' growth and the formation of photosynthetic apparatus. The mechanism by which soil overlay drives morphogenetic changes in plants, however, remains poorly understood, particularly with regard to the means by which the cellular processes of different organs are coordinated in response to disparate soil conditions. Here, we illustrate that the soil overlay quantitatively activates seedlings' ethylene production, and an EIN3/EIN3-like 1-dependent ethylene-response cascade is required for seedlings to successfully emerge from the soil. Under soil, an ERF1 pathway is activated in the hypocotyl to slow down cell elongation, whereas a PIF3 pathway is activated in the cotyledon to control the preassembly of photosynthetic machinery. Moreover, this latter PIF3 pathway appears to be coupled to the ERF1-regulated upward-growth rate. The coupling of these two pathways facilitates the synchronized progression of etioplast maturation and hypocotyl growth, which, in turn, ultimately enables seedlings to maintain the amount of protochlorophyllide required for rapid acquisition of photoautotrophic capacity without suffering from photooxidative damage during the dark-to-light transition. Our findings illustrate the existence of a genetic signaling pathway driving soil-induced plant morphogenesis and define the specific role of ethylene in orchestrating organ-specific soil responses in Arabidopsis seedlings.

Effect of low temperature on chlorophyll biosynthesis in albinism line of wheat (Triticum aestivum) FA85.

The 'stage albinism line of winter wheat' FA85 exhibits a severe block in chlorophyll (Chl) biosynthesis with prolonged low-temperature treatment. The correlations between leaf color and low temperature provide more comprehensive understanding of low temperature as an environmental signal that regulate the metabolic changes in the entire Chl-synthesizing pathway. In this study, we investigated differences in Chl biosynthesis between leaves of Aibian1 and FA85 by measuring their Chl precursors and heme content, transcripts for key genes of Chl biosynthesis and key enzyme activities. With prolonged low-temperature treatment, the Chl content gradually decreased, but Chl precursors, including protoporphyrin IX, Mg-protoporphyrin IX and protochlorophyllide (Pchlide), simultaneously accumulated. Parallel to the decline in Chl content, the protoporphyrin IX distribution toward Chl synthesis was less than that in heme synthesis in the leaves of FA85. Corresponding to the change of protoporphyrin IX distribution, the relative changes in magnesium chelatase (EC 6.6.1.1) and ferrochelatase (EC 4.99.1.1) activities in the leaves of FA85 also indirectly reflected channeling of the metabolic flow into heme rather than Chl. A drastic loss in the transcripts for Pchlide oxidoreductase (EC 1.3.1.33) and Chl synthase (EC 2.5.1.62) accounted for a decrease in the metabolic flux and the re-direction of metabolites. The high-level accumulations of Chl precursors and traces of Chl in the leaves of FA85 suggest that a severe block between the steps from Pchlide to Chl formation during Chl biosynthesis is partially derived from the transcriptional downregulation of Pchlide oxidoreductase and Chl synthase.

The SCO2 protein disulphide isomerase is required for thylakoid biogenesis and interacts with LHCB1 chlorophyll a/b binding proteins which affects chlorophyll biosynthesis in Arabidopsis seedlings.

The process of chloroplast biogenesis requires a multitude of pathways and processes to establish chloroplast function. In cotyledons of seedlings, chloroplasts develop either directly from proplastids (also named eoplasts) or, if germinated in the dark, via etioplasts, whereas in leaves chloroplasts derive from proplastids in the apical meristem and are then multiplied by division. The snowy cotyledon 2, sco2, mutations specifically disrupt chloroplast biogenesis in cotyledons. SCO2 encodes a chloroplast-localized protein disulphide isomerase, hypothesized to be involved in protein folding. Analysis of co-expressed genes with SCO2 revealed that genes with similar expression patterns encode chloroplast proteins involved in protein translation and in chlorophyll biosynthesis. Indeed, sco2-1 accumulates increased levels of the chlorophyll precursor, protochlorophyllide, in both dark grown cotyledons and leaves. Yeast two-hybrid analyses demonstrated that SCO2 directly interacts with the chlorophyll-binding LHCB1 proteins, being confirmed in planta using bimolecular fluorescence complementation (BIFC). Furthermore, ultrastructural analysis of sco2-1 chloroplasts revealed that formation and movement of transport vesicles from the inner envelope to the thylakoids is perturbed. SCO2 does not interact with the signal recognition particle proteins SRP54 and FtsY, which were shown to be involved in targeting of LHCB1 to the thylakoids. We hypothesize that SCO2 provides an alternative targeting pathway for light-harvesting chlorophyll binding (LHCB) proteins to the thylakoids via transport vesicles predominantly in cotyledons, with the signal recognition particle (SRP) pathway predominant in rosette leaves. Therefore, we propose that SCO2 is involved in the integration of LHCB1 proteins into the thylakoids that feeds back on the regulation of the tetrapyrrole biosynthetic pathway and nuclear gene expression.

Methods for analysis of photosynthetic pigments and steady-state levels of intermediates of tetrapyrrole biosynthesis.

Tetrapyrroles and carotenoids are required for many indispensable functions in photosynthesis. Tetrapyrroles are essential metabolites for photosynthesis, redox reaction, and detoxification of reactive oxygen species and xenobiotics, while carotenoids function as accessory pigments, in photoprotection and in attraction to animals. Their branched metabolic pathways of synthesis and degradation are tightly controlled to provide adequate amounts of each metabolite (carotenoids/tetrapyrroles) and to prevent accumulation of photoreactive intermediates (tetrapyrroles). Many Arabidopsis mutants and transgenic plants have been reported to show variations in steady-state levels of tetrapyrrole intermediates and contents of different carotenoid species. It is a challenging task to determine the minute amounts of these metabolites to assess the metabolic flow and the activities of both pigment-synthesising and degrading pathways, to unravel limiting enzymatic steps of these biosynthetic pathways, and to characterise mutants with accumulating intermediates. In this chapter, we present a series of methods to qualify and quantify anabolic and catabolic intermediates of Arabidopsis tetrapyrrole metabolism, and describe a common method for quantification of different plant carotenoid species. Additionally, we introduce two methods for quantification of non-covalently bound haem. The approach of analysing steady-state levels of tetrapyrrole intermediates in plants, when applied in combination with analyses of transcripts, proteins, and enzyme activities, enables the biochemical and genetic elucidation of the tetrapyrrole pathway in wild-type plants, varieties, and mutants. Steady-state levels of tetrapyrrole intermediates are only up to 1/1,000 of the amounts of the accumulating end-products, chlorophyll, and haem. Although present in very low amounts, the accumulation and availability of tetrapyrrole intermediates have major consequences on the physiology and activity of chloroplasts due to their additional photoreactive and possible signalling functions. Although adjusted for Arabidopsis tetrapyrrole metabolites, the presented methods can also be applied for analysis of cyanobacterial and other plant tetrapyrroles.

Rapid dark repression of 5-aminolevulinic acid synthesis in green barley leaves.

In photosynthetic organisms chlorophyll and heme biosynthesis is tightly regulated at various levels in response to environmental adaptation and plant development. The formation of 5-aminolevulinic acid (ALA) is the key regulatory step and provides adequate amounts of the common precursor molecule for the Mg and Fe branches of tetrapyrrole biosynthesis. Pathway control prevents accumulation of metabolic intermediates and avoids photo-oxidative damage. In angiosperms reduction of protochlorophyllide (Pchlide) to chlorophyllide is catalyzed by the light-dependent NADPH:Pchlide oxidoreductase (POR). Although a correlation between down-regulated ALA synthesis and accumulation of Pchlide in the dark was proposed a long time ago, the time-resolved mutual dependency has never been analyzed. Taking advantage of the high metabolic activity of young barley (Hordeum vulgare L.) seedlings, in planta ALA synthesis could be determined with high time-resolution. ALA formation declined immediately after transition from light to dark and correlated with an immediate accumulation of POR-bound Pchlide within the first 60 min in darkness. The flu homologous barley mutant tigrina d(12) uncouples ALA synthesis from dark-suppression and continued to form ALA in darkness without a significant change in synthesis rate in this time interval. Similarly, inhibition of protoporphyrinogen IX oxidase by acifluorfen resulted in a delayed accumulation of Pchlide during the entire dark period and a weak repression of ALA synthesis in darkness. Moreover, it is demonstrated that dark repression of ALA formation relies rather on rapid post-translational regulation in response to accumulating Pchlide than on changes in nuclear gene expression.

[Light-induced reduction of protochlorophyllide in angiosperms and chloroplast development]. / Indukowana swiatlem redukcja protochlorofilidu u okrytonasiennych a rozwój chloroplastów.

One of the final reactions of chlorophyll (Chl) biosynthesis, e.g: photoreduction of protochlorophyllide (Pchlid) to chlorophyllide (Chlid) is a light-induced process in Angiosperm plants and it is catalyzed by light-dependent NADPH-Pchlid oxidoreductase (1.3.1.33; LPOR). In darkness, Chl biosynthesis is stopped at the stage of Pchlid formation. Seedlings and plastids develop according to a different pattern than that observed in the light. Moreover, synthesis of some proteins of the photosynthetic apparatus is inhibited. Light triggers the Pchlid photoreduction to Chlid, which induces the cascade of biochemical reactions and structural changes leading to the assembly of thylakoid membranes. In the present paper, the current knowledge on LPOR protein, mechanism of Pchlid to Chlid photoreduction, the role of lipid structure in etioplasts as well as spectral properties of Pchlid in etiolated seedlings and model systems is summarized.

A novel insight into the regulation of light-independent chlorophyll biosynthesis in Larix decidua and Picea abies seedlings.

Light-independent chlorophyll (Chl) biosynthesis is a prerequisite for the assembly of photosynthetic pigment-protein complexes in the dark. Dark-grown Larix decidua Mill. seedlings synthesize Chl only in the early developmental stages and their Chl level rapidly declines during the subsequent development. Our analysis of the key regulatory steps in Chl biosynthesis revealed that etiolation of initially green dark-grown larch cotyledons is connected with decreasing content of glutamyl-tRNA reductase and reduced 5-aminolevulinic acid synthesizing capacity. The level of the Chl precursor protochlorophyllide also declined in the developing larch cotyledons. Although the genes chlL, chlN and chlB encoding subunits of the light-independent protochlorophyllide oxidoreductase were constitutively expressed in the larch seedlings, the accumulation of the ChlB subunit was developmentally regulated and ChlB content decreased in the fully developed cotyledons. The efficiency of chlB RNA-editing was also reduced in the mature dark-grown larch seedlings. In contrast to larch, dark-grown seedlings of Picea abies (L.) Karst. accumulate Chl throughout their whole development and show a different control of ChlB expression. Analysis of the plastid ultrastructure, photosynthetic proteins by Western blotting and photosynthetic parameters by gas exchange and Chl fluorescence measurements provide additional experimental proofs for differences between dark and light Chl biosynthesis in spruce and larch seedlings.

Identification of a novel vinyl reductase gene essential for the biosynthesis of monovinyl chlorophyll in Synechocystis sp. PCC6803.

The vast majority of oxygenic photosynthetic organisms use monovinyl chlorophyll for their photosynthetic reactions. For the biosynthesis of this type of chlorophyll, the reduction of the 8-vinyl group that is located on the B-ring of the macrocycle is essential. Previously, we identified the gene encoding 8-vinyl reductase responsible for this reaction in higher plants and termed it DVR. Among the sequenced genomes of cyanobacteria, only several Synechococcus species contain DVR homologues. Therefore, it has been hypothesized that many other cyanobacteria producing monovinyl chlorophyll should contain a vinyl reductase that is unrelated to the higher plant DVR. To identify the cyanobacterial gene that is responsible for monovinyl chlorophyll synthesis, we developed a bioinformatics tool, correlation coefficient calculation tool, which calculates the correlation coefficient between the distributions of a certain phenotype and genes among a group of organisms. The program indicated that the distribution of a gene encoding a putative dehydrogenase protein is best correlated with the distribution of the DVR-less cyanobacteria. We subsequently knocked out the corresponding gene (Slr1923) in Synechocystis sp. PCC6803 and characterized the mutant. The knock-out mutant lost its ability to synthesize monovinyl chlorophyll and accumulated 3,8-divinyl chlorophyll instead. We concluded that Slr1923 encodes the vinyl reductase or a subunit essential for monovinyl chlorophyll synthesis. The function and evolution of 8-vinyl reductase genes are discussed.

Characterization of the Arabidopsis thaliana mutant pcb2 which accumulates divinyl chlorophylls.

We characterized the pcb2 (pale-green and chlorophyll b reduced 2) mutant. We found through electron microscopic observation that chloroplasts of pcb2 mesophyll cells lacked distinctive grana stacks. High-performance liquid chromatography (HPLC) analysis showed that the pcb2 mutant accumulated divinyl chlorophylls, and the relative amount of divinyl chlorophyll b was remarkably less than that of divinyl chlorophyll a. The responsible gene was mapped in an area of 190 kb length at the upper arm of the 5th chromosome, and comparison of DNA sequences revealed a single nucleotide substitution causing a nonsense mutation in At5g18660. Complementation analysis confirmed that the wild-type of this gene suppressed the phenotypes of the mutation. Antisense transformants of the gene also accumulated divinyl chlorophylls. The genes homologous to At5g18660 are conserved in a broad range of species in the plant kingdom, and have similarity to reductases. Our results suggest that the PCB2 product is divinyl protochlorophyllide 8-vinyl reductase.

The jasmonate-free rice mutant hebiba is affected in the response of phyA'/phyA" pools and protochlorophyllide biosynthesis to far-red light.

Phytochrome (phy) A in its two native isoforms (phyA' and phyA") and the active (Pchlide(655)) and inactive (Pchlide(633)) protochlorophyllides were investigated by low-temperature fluorescence spectroscopy in the tips of rice (Oryza sativa L. Japonica cv Nihonmasari) coleoptiles from wild type (WT) and the jasmonate-deficient mutant hebiba. The seedlings were either grown in the dark or under pulsed (FRp) or continuous (FRc) far-red light (lambda(a) >/= 720 nm) of equal fluences. In the dark, the mutant had a long mesocotyl and a short coleoptile, whereas the situation was reversed under FR: short mesocotyl and long coleoptile, suggesting that the effect is mediated by phyA. Under these conditions the WT displayed a short coleoptile and emergence of the first leaf. In the dark, the spectroscopic and photochemical properties of phyA, its content and the proportion of its two pools, phyA' and phyA", were virtually identical between WT and hebiba. However, the total content of protochlorophyllides was higher in the mutant. Upon illumination with FRc, [phyA] declined in the WT and the ratio between phyA' and phyA" shifted towards phyA". In hebiba, the light-induced decline of [phyA] was less pronounced and the ratio between phyA' and phyA" did not shift. Moreover, in the WT, FRp stimulated the biosynthesis of Pchlide(655), whereas FRc was inhibiting. In contrast, in the mutant, both FRp and FRc stimulated the synthesis of Pchlide(655). This means that FRc caused the opposite effect in hebiba. This difference correlates with a slower photodestruction of primarily the light-labile phyA' pool in hebiba.

Up-regulation by phytochrome A of the active protochlorophyllide, Pchlide655, biosynthesis in dicots under far-red light.

It is well-documented that phytochrome A (phyA) down-regulates the synthesis of NADPH:protochlorophyllide (Pchlide) oxidoreductase and active Pchlide(655) under far-red light (FR). In this work, we demonstrate that phyA can up-regulate the synthesis of Pchlide(655) under FR as well and that its sign and extent depend on plant species and tissue. With the use of fluorescence spectroscopy, it was found that [Pchlide(655)] in the upper stems of FR-grown seedlings of pea and tobacco increased > or =10-fold and much lower in cotyledons or leaves as compared with the dark-grown. In the upper stems of Arabidopsis and tomato, the positive effect of FR was low, 1.2- to 1.5-fold, and the negative effect of FR was seen in cotyledons. In stems of wild-type (WT) tobacco and its line overexpressing full-length oat phyA (FL), we observed gross stimulating effect of FR while in its line overexpressing N-terminally truncated (Delta7-69) oat phyA (NA) it was low. Because WT and FL comprise both native phyA forms, phyA' and phyA", while NA, only phyA", the regulation under FR can be associated with phyA', while phyA" inhibits the action of phyA'. In etiolated seedlings of the NA line, [Pchlide(655)] was much higher than in those of WT and FL suggesting that phyA" may have relation to this enhancement. The regulation of Pchlide(633) in contrast to Pchlide(655) was positive independent of the plant species and tissue.

Arabidopsis CHL27, located in both envelope and thylakoid membranes, is required for the synthesis of protochlorophyllide.

CHL27, the Arabidopsis homologue to Chlamydomonas Crd1, a plastid-localized putative diiron protein, is required for the synthesis of protochlorophyllide and therefore is a candidate subunit of the aerobic cyclase in chlorophyll biosynthesis. delta-Aminolevulinic acid-fed antisense Arabidopsis plants with reduced amounts of Crd1/CHL27 accumulate Mg-protoporphyrin IX monomethyl ester, the substrate of the cyclase reaction. Mutant plants have chlorotic leaves with reduced abundance of all chlorophyll proteins. Fractionation of Arabidopsis chloroplast membranes shows that Crd1/CHL27 is equally distributed on a membrane-weight basis in the thylakoid and inner-envelope membranes.