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Sulfide plays essential roles in controlling various physiological activities in almost all organisms. Although recent evidence has demonstrated that sulfide is endogenously generated and metabolized into polysulfides inside the cells, the relationship between polysulfide metabolism and polysulfide-sensing mechanisms is not well understood. To better define this interplay between polysulfide metabolism and sensing in cells, we investigated the role of polysulfide-metabolizing enzymes such as sulfide:quinone oxidoreductase (SQR) on the temporal dynamics of cellular polysulfide speciation and on the transcriptional regulation by the persulfide-responsive transcription factor SqrR in Rhodobacter capsulatus. We show that disruption of the sqr gene resulted in the loss of SqrR repression by exogenous sulfide at longer culture times, which impacts the speciation of intracellular polysulfides of Δsqr vs. wild-type strains. Both the attenuated response of SqrR and the change in polysulfide dynamics of the Δsqr strain is fully reversed by the addition to cells of cystine-derived polysulfides, but not by glutathione disulfide (GSSG)-derived polysulfides. Furthermore, cysteine persulfide (CysSSH) yields a higher rate of oxidation of SqrR relative to glutathione persulfide (GSSH), which leads to DNA dissociation in vitro. The oxidation of SqrR was confirmed by a mass spectrometry-based kinetic profiling strategy that showed distinct polysulfide-crosslinked products obtained with CysSSH vs. GSSH. Taken together, these results establish a novel association between the metabolism of polysulfides and the mechanisms for polysulfide sensing inside the cells.

Significance The signaling pathway between chloroplasts and the nucleus (retrograde signaling) is important for the correct development of the photosynthetic apparatus of plant seedlings. The pathway is still not understood, but the majority of mutants with altered signaling (gun mutants) implicate the tetrapyrrole molecule heme in this process. In this article, we have demonstrated that the major retrograde signaling protein GUN1 can bind tetrapyrroles and regulate the flow through the tetrapyrrole biosynthesis pathway. The results support a role for tetrapyrroles in mediating retrograde signaling and open up the opportunity to develop a unifying hypothesis for this pathway that takes account of all identified gun mutants., The biogenesis of the photosynthetic apparatus in developing seedlings requires the assembly of proteins encoded on both nuclear and chloroplast genomes. To coordinate this process there needs to be communication between these organelles, but the retrograde signals by which the chloroplast communicates with the nucleus at this time are still essentially unknown. The Arabidopsis thaliana genomes uncoupled (gun) mutants, that show elevated nuclear gene expression after chloroplast damage, have formed the basis of our understanding of retrograde signaling. Of the 6 reported gun mutations, 5 are in tetrapyrrole biosynthesis proteins and this has led to the development of a model for chloroplast-to-nucleus retrograde signaling in which ferrochelatase 1 (FC1)-dependent heme synthesis generates a positive signal promoting expression of photosynthesis-related genes. However, the molecular consequences of the strongest of the gun mutants, gun1, are poorly understood, preventing the development of a unifying hypothesis for chloroplast-to-nucleus signaling. Here, we show that GUN1 directly binds to heme and other porphyrins, reduces flux through the tetrapyrrole biosynthesis pathway to limit heme and protochlorophyllide synthesis, and can increase the chelatase activity of FC1. These results raise the possibility that the signaling role of GUN1 may be manifested through changes in tetrapyrrole metabolism, supporting a role for tetrapyrroles as mediators of a single biogenic chloroplast-to-nucleus retrograde signaling pathway.

Etioplasts developed in angiosperm cotyledon cells in darkness rapidly differentiate into chloroplasts with illumination. This process involves dynamic transformation of internal membrane structures from the prolamellar bodies (PLBs) and prothylakoids (PTs) in etioplasts to thylakoid membranes in chloroplasts. Although two galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), are predominant lipid constituents of membranes in both etioplasts and chloroplasts, their roles in the structural and functional transformation of internal membranes during etioplast-to-chloroplast differentiation are unknown. We previously reported that a 36% loss of MGDG by an artificial microRNA targeting major MGDG synthase (amiR-MGD1) only slightly affected PLB structures but strongly impaired PT formation and protochlorophyllide biosynthesis. Meanwhile, strong DGDG deficiency in a DGDG synthase mutant (dgd1) disordered the PLB lattice structure in addition to impaired PT development and protochlorophyllide biosynthesis. In this study, thylakoid biogenesis after PLB disassembly with illumination was strongly perturbed by amiR-MGD1. The amiR-MGD1 expression impaired the accumulation of Chl and the major light-harvesting complex II protein (LHCB1), which may inhibit rapid transformation from disassembled PLBs to the thylakoid membrane. As did amiR-MGD1 expression, dgd1 mutation impaired the accumulation of Chl and LHCB1 during etioplast-to-chloroplast differentiation. Furthermore, unlike in amiR-MGD1 seedlings, in dgd1 seedlings, disassembly of PLBs after illumination was retarded. Because DGDG but not MGDG prefers to form the bilayer lipid phase in membranes, the MGDG-to-DGDG ratio may strongly affect the transformation of PLBs to the thylakoid membrane during etioplast-to-chloroplast differentiation.

Reactive sulfur species (RSS) are involved in bioactive regulation via persulfidation of proteins. However, how cells regulate RSS-based signaling and RSS metabolism is poorly understood, despite the importance of universal regulation systems in biology. We previously showed that the persulfide-responsive transcriptional factor SqrR acts as a master regulator of sulfide-dependent photosynthesis in proteobacteria. Here, we demonstrated that SqrR also binds heme at a near one-to-one ratio with a binding constant similar to other heme-binding proteins. Heme does not change the DNA-binding pattern of SqrR to the target gene promoter region; however, DNA-binding affinity of SqrR is reduced by the binding of heme, altering its regulatory activity. Circular dichroism spectroscopy clearly showed secondary structural changes in SqrR by the heme binding. Incremental change in the intracellular heme concentration is associated with small, but significant reduction in the transcriptional repression by SqrR. Overall, these results indicate that SqrR has an ability to bind heme to modulate its DNA-binding activity, which may be important for the precise regulation of RSS metabolism in vivo.

Supplemental Fig. 1, Fig. 2, Fig. 3, Fig. 4, Table 1

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.

The biogenesis of the photosynthetic apparatus in developing chloroplasts requires the assembly of proteins encoded on both nuclear and chloroplast genomes1. To co-ordinate this process there needs to be communication between these organelles, and while we have a good understanding of how the nucleus controls chloroplast development, how the chloroplast communicates with the nucleus at this time is still essentially unknown2. What we do know comes from pioneering work in which a series of genomes uncoupled (gun) mutants were identified that show elevated nuclear gene expression after chloroplast damage3. Of the six reported gun mutations, five are in tetrapyrrole biosynthesis proteins4-6 and this has led to the development of a model for chloroplast-to-nucleus retrograde signaling in which ferrochelatase 1 (FC1)-dependent heme synthesis generates a positive signal promoting expression of photosynthesis-related genes6. However, the molecular consequences of the strongest of the gun mutants, gun17, is unknown, preventing the development of a unifying hypothesis for chloroplast-to-nucleus signaling. Here, we show that GUN1 directly binds to heme and other metal-porphyrins, affects flux through the tetrapyrrole biosynthesis pathway and can increase the chelatase activity of FC1. These results raise the possibility that the signaling role of GUN1 may be manifested through changes in tetrapyrrole metabolism and supports a role for tetrapyrroles as mediators of a single biogenic chloroplast-to-nucleus retrograde signaling pathway.

Oxygenic photosynthetic organisms synthesize chlorophyll (Chl), the photoreactive pigment functioning in light-harvesting for photosynthesis, via multiple enzymatic steps, which belong to a common metabolic pathway of tetrapyrroles, such as heme, siroheme and phytochromobiline. To coordinate Chl biosynthesis with the formation of pigment-binding proteins of the photosynthetic complexes and to prevent photodamage by free Chl and its accumulating photoreactive precursors, photosynthetic organisms have developed various regulatory mechanisms. Among multiple levels of control, transcriptional regulation of genes comprising the Chl biosynthesis pathway plays a crucial role in fine-tuning the metabolic flow of tetrapyrroles with the varying needs of Chl under various developmental and environmental conditions. As mainly explored in Arabidopsis thaliana, expression of plant key Chl biosynthesis genes is tightly coordinated with other photosynthesis-associated nuclear genes in response to light and other stimuli, including the circadian clock, strictly organized under developmental programs, and immediately shut off when chloroplasts become dysfunctional. Various transcription factors associated with light-signaling, hormonal regulation, and developmental and nutritional responses function in a complex manner to orchestrate Chl biosynthesis with the development and maintenance of chloroplasts. This chapter summarizes transcriptional profiles of Chl biosynthesis genes in Arabidopsis and their regulation through several transcription factors and associated regulators in response to environmental and endogenous cues.

Chloroplast biogenesis involves the coordinated expression of the plastid and nuclear genomes, requiring information to be sent from the nucleus to the developing chloroplasts and vice versa. Although it is well known how the nucleus controls chloroplast development, it is still poorly understood how the plastid communicates with the nucleus. Currently, haem is proposed as a plastid-to-nucleus (retrograde) signal that is involved in various physiological regulations, such as photosynthesis-associated nuclear genes expression and cell cycle in plants and algae. However, components that transduce haem-dependent signalling are still unidentified. In this study, by using haem-immobilized high-performance affinity beads, we performed proteomic analysis of haem-binding proteins from Arabidopsis thaliana and Cyanidioschyzon merolae . Most of the identified proteins were non-canonical haemoproteins localized in various organelles. Interestingly, half of the identified proteins were nucleus proteins, some of them have a similar function or localization in either or both organisms. Following biochemical analysis of selective proteins demonstrated haem binding. This study firstly demonstrates that nucleus proteins in plant and algae show haem-binding properties. This article is part of the theme issue ‘Retrograde signalling from endosymbiotic organelles’.

Angiosperms germinated in the dark develop etioplasts, the chloroplast precursors, in cotyledon cells. Etioplasts contain lattice membrane structures called prolamellar bodies (PLBs) and lamellar prothylakoids as internal membrane systems. PLBs accumulate the chlorophyll intermediate protochlorophyllide (Pchlide) in a complex with NADPH and light-dependent NADPH:Pchlide oxidoreductase (LPOR). Two galactolipids, monogalactosyldiacylglycerol and digalactosyldiacylglycerol (DGDG), are major constituents of etioplast membranes. We previously reported that monogalactosyldiacylglycerol facilitates the synthesis of Pchlide and the formation of the Pchlide-LPOR-NADPH complex in etioplasts, but the importance of DGDG in etioplasts is still unknown. To determine the role of DGDG in etioplast development and functions, we characterized a knockout mutant (dgd1) of Arabidopsis (Arabidopsis thaliana) DGD1, which encodes the major isoform of DGDG synthase, in the etioplast development stage. In etiolated dgd1 seedlings, DGDG content decreased to 20% of the wild-type level, the lattice structure of PLBs was disordered, and the development of prothylakoids was impaired. In addition, membrane-associated processes of Pchlide biosynthesis, formation of the Pchlide-LPOR-NADPH complex, and dissociation of the complex after the photoconversion of Pchlide to chlorophyllide were impaired in dgd1, although the photoconversion reaction by LPOR was not affected by the DGDG deficiency. Total carotenoid content also decreased in etiolated dgd1 seedlings, but the carotenoid composition was unchanged. Our data demonstrate a deep involvement of DGDG in the formation of the internal membrane structures in etioplasts as well as in membrane-associated processes of pigment biosynthesis and pigment-protein complex organization.

Tetrapyrroles such as heme and chlorophyll are essential for biological processes, including oxygenation, respiration, and photosynthesis. In the tetrapyrrole biosynthesis pathway, protoporphyrinogen IX oxidase (Protox) catalyzes the formation of protoporphyrin IX, the last common intermediate for the biosynthesis of heme and chlorophyll. Three nonhomologous isofunctional enzymes, HemG, HemJ, and HemY, for Protox have been identified. To reveal the distribution and evolution of the three Protox enzymes, we identified homologs of each along with other heme biosynthetic enzymes by whole-genome clustering across three domains of life. Most organisms possess only one of the three Protox types, with some exceptions. Detailed phylogenetic analysis revealed that HemG is mostly limited to γ-Proteobacteria whereas HemJ may have originated within α-Proteobacteria and transferred to other Proteobacteria and Cyanobacteria. In contrast, HemY is ubiquitous in prokaryotes and is the only Protox in eukaryotes, so this type may be the ancestral Protox. Land plants have a unique HemY homolog that is also shared by Chloroflexus species, in addition to the main HemY homolog originating from Cyanobacteria. Meanwhile, organisms missing any Protox can be classified into two groups; those lacking most heme synthetic genes, which necessarily depend on external heme supply, and those lacking only genes involved in the conversion of uroporphyrinogen III into heme, which would use a precorrin2-dependent alternative pathway. However, hemN encoding coproporphyrinogen IX oxidase was frequently found in organisms lacking Protox enzyme, which suggests a unique role of this gene other than in heme biosynthesis.

The development of plant chloroplasts is regulated by various developmental, environmental, and hormonal cues. In Arabidopsis (Arabidopsis thaliana), chloroplast development is repressed in roots via auxin signaling. However, roots develop chloroplasts when they are detached from the shoot. In contrast to auxin, cytokinin positively affects chloroplast development in roots, but the role and signaling pathway of cytokinin in the root greening response remain unclear. To understand the regulatory pathways of chloroplast development in the plant stress response, we examined the mechanisms underlying the conditional greening of detached roots. In wild-type Arabidopsis roots, shoot removal activates type B ARABIDOPSIS RESPONSE REGULATOR (ARR)-mediated cytokinin signaling and induces chlorophyll accumulation and photosynthetic remodeling. ARR1 and ARR12 are essential for up-regulating nucleus- and plastid-encoded genes associated with chloroplast development in detached roots. In this process, WOUND INDUCED DEDIFFERENTIATION1 and class B GATA transcription factors (B-GATAs) act upstream and downstream of ARRs, respectively. Overexpression of B-GATAs promotes root greening, as does shoot removal, dependent on a light signaling transcription factor, LONG HYPOCOTYL5. Auxin represses the root greening response independent of ARR signaling. GNC-LIKE (GNL), a B-GATA, is strongly up-regulated in detached roots via ARR1 and ARR12 but is repressed by auxin, so GNL may function at the point of convergence of cytokinin and auxin signaling in the root greening response.

Photosynthetic organisms contain some form of the light-absorbing pigment chlorophyll (Chl). Photosynthetic organisms that perform oxygenic photosynthesis, such as cyanobacteria, algae, and plants, synthesize Chl a. A group of cyanobacteria (prochlorophytes), green algae, and plants also contain Chl b. Photosynthetic bacteria, which perform anaerobic photosynthesis, produce a variety of derivatives called bacteriochlorophylls (Bchls). In the order of their discovery, Chls and Bchls are named a–f and a–g, respectively. Chls contain a tetrapyrrole ring structure like that found in heme (Figure 9.1). However, Chls bind an Mg atom in the center of its ring structure, while hemes bind an Fe atom. In addition, a hydrophobic phytol chain is attached to the ring structure of Chl that makes the molecule extremely nonpolar. All Chls are noncovalently bound to various photosystem subunits in the thylakoid membrane.

The Arabidopsis thaliana L. SOUL/haem-binding proteins, AtHBPs belong to a family of five members. The Arabidopsis cytosolic AtHBP1 (At1g17100) and AtHBP2 (At2g37970) have been shown to bind porphyrins and metalloporphyrins including haem. In contrast to the cytosolic localization of these haem-binding proteins, AtHBP5 (At5g20140) encodes a protein with an N-terminal transit peptide that probably directs targeting to the chloroplast. In this report, it is shown that AtHBP5 binds haem and interacts with the haem oxygenase, HY1, in both yeast two-hybrid and BiFC assays. The expression of HY1 is repressed in the athbp5 T-DNA knockdown mutant and the accumulation of H(2)O(2) is observed in athbp5 seedlings that are treated with methyl jasmonate (MeJA), a ROS-producing stress hormone. In contrast, AtHBP5 over-expressing plants show a decreased accumulation of H(2)O(2) after MeJA treatment compared with the controls. It is proposed that the interaction between the HY1 and AtHBP5 proteins participate in an antioxidant pathway that might be mediated by reaction products of haem catabolism.

Regulation of chlorophyll metabolism comprises strong transcriptional control together with a range of post-translational mechanisms during chloroplast biogenesis. Recently we reported that chlorophyll biosynthesis in Arabidopsis thaliana roots is regulated by auxin/cytokinin signaling via the combination of two transcription factors, LONG-HYPOCOTYL5 (HY5) and GOLDEN2-LIKE2 (GLK2). In this study, we examined the involvement of cis-elements in the expression of chlorophyll biosynthesis genes. Searches for predicted cis-elements in key chlorophyll biosynthesis genes and their co-expressed genes revealed coexistence of the G-box motif and the CCAATC motif, which may be targeted by HY5 and GLK factors, respectively, in their promoter regions. Deletion of the G-box from the promoter of the CHLH gene encoding the H subunit of Mg-chelatase resulted in the absence of its expression in roots but not in shoots, showing a differing involvement of the G-box in CHLH expression between shoots and roots. Our data suggest that transcription factors and cis-elements participating chlorophyll biosynthesis are substantially changed during organ differentiation, which may be linked to the differentiation of plastids.

Tetrapyrroles such as chlorophyll and heme are co-factors for essential proteins involved in a wide variety of crucial cellular functions. Nearly 2% of the proteins encoded by the Arabidopsis thaliana genome are thought to bind tetrapyrroles, demonstrating their central role in plant metabolism. Although the enzymes required for tetrapyrrole biosynthesis are well characterized, there are still major questions about the regulation of the pathway, and the transport of tetrapyrroles within cells. These issues are important, as misregulation of tetrapyrrole metabolism can lead to severe photo-oxidative stress, and because tetrapyrroles have been implicated in signaling pathways coordinating interactions between plant organelles. In this review, we discuss the cell biology of tetrapyrrole metabolism and its implications for tetrapyrroles as signaling molecules.

SummaryMono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively) constitute the bulk of membrane lipidsin plant chloroplasts. Mutant analyses in Arabidopsis have shown that these galactolipids are essential forchloroplast biogenesis and photoautotrophic growth. Moreover, these non-phosphorous lipids are proposedto participate in low-phosphate (Pi) adaptations. Under Pi-limited conditions, a drastic accumulation of DGDGoccurs concomitantly with a large reduction in membrane phospholipids, suggesting that plants substituteDGDG for phospholipids during Pi starvation. Previously, we reported that among the three MGDG synthasegenes (MGD1, MGD2and MGD3), the type-B MGD2and MGD3are upregulated in parallel with DGDG synthasegenes during Pi starvation. Here, we describe the identification and characterization of T-DNA insertionalmutants of Arabidopsis type-B MGD genes. Under Pi-starved conditions, the mgd3-1 mutant showed a drasticreductioninDGDGaccumulation,particularlyintheroot,indicatingthatMGD3isthemainisoformresponsiblefor DGDG biosynthesis in Pi-starved roots. Moreover, in the roots of mgd2 mgd3 plants, Pi stress-inducedaccumulation of DGDG was almost fully abolished, showing that type-B MGD enzymes are essential formembrane lipid remodeling in Pi-starved roots. Reductions in fresh weight, root growth and photosyntheticperformancewere alsoobservedinthesemutants underPi-starvedconditions.Theseresults demonstratethatPi stress-induced membrane lipid remodeling is important in plant growth during Pi starvation. Thewidespread distribution of type-B MGD genes in land plants suggests that membrane lipid remodelingmediated by type-B MGD enzymes is a potent adaptation to Pi deficiency for land plants.Keywords: galactolipid, phosphate starvation, monogalactosyldiacylglycerol, MGDG synthase, digalactosyl-diacylglycerol.IntroductionLife in all organisms relies on the presence of lipid bilayercell membranes, which separate the interior of cells fromtheir environment. In the plastids of higher plants, the non-phosphorous galactolipids monogalactosyldiacylglycerol(MGDG)anddigalactosyldiacylglycerol(DGDG)arethemainconstituents of the membrane lipids, whereas phospholip-ids are the predominant components of other subcellularmembranes, such as the plasma membrane, the endoplas-mic reticulum (ER) and the mitochondrial membranes(Joyard et al., 1998). In chloroplasts, MGDG and DGDG

The first step of chlorophyll biosynthesis is catalyzed by a Mg-chelatase composed of the subunits CHLI, CHLD and CHLH. Mg-chelatase requires ATP hydrolysis that can be attributed to CHLI. Arabidopsis has two CHLI isoforms, CHLI1 and CHLI2, that have similar expression profiles, but it has been suggested that CHLI2 has limited function in the Mg-chelatase complex. Recently, we showed that Arabidopsis CHLI1 is an ATPase and a target of chloroplast thioredoxin. Here, we demonstrate that CHLI2 also has ATPase activity but with a lower Vmax and higher Km ATP than CHLI1. We confirmed the thioredoxin-dependent reduction of a disulfide bond in CHLI2 and thiol-modulation of its ATPase activity. We then examined the physiological contribution of CHLI2 using a chli2 T-DNA knockout line. Although visible phenotype of homozygous chli2 mutants was almost comparable to wild type, the mutant accumulated significantly less chlorophyll. Furthermore, cs/cs; chli2/chli2 double mutants were almost albino. There were three phenotypes among progenies segregated from the cs/cs; CHLI2/chli2 parent: cs-like pale green, yellow, and almost albino were obtained in the approximate ratio of 1:2:0.7. PCR analysis confirmed that the chli2 mutation is semidominant on a homozygous cs background. These results reveal that although CHLI2 plays a limited role in chlorophyll biosynthesis, this subunit certainly contributes to the assembly of the Mg-chelatase complex.

The plastid plays a vital role in various cellular activities within plant cells including photosynthesis and other metabolic pathways. It is believed that the functional status of the plastid is somehow monitored by the nucleus to optimize the expression of genes encoding plastid proteins. The currently dominant model for plastid-derived signaling (“plastid signaling”) proposes that Mg-protoporphyrin IX (MgProto) is a negative signal that represses the expression of a wide range of nuclear genes encoding plastid-localized proteins when plastid development is inhibited. In this study, we have re-evaluated this hypothesis by quantifying the steady-state levels of MgProto (as well as its neighboring intermediates protoporphyrin IX and Mg-Proto monomethyl ester [MgProtoMe]) in Arabidopsis plants with altered plastid signaling responses as monitored by expression of the Lhcb1 , RBCS , HEMA1 , BAM3 and CA1 genes. In addition, we have examined the correlation between gene expression and MgProto (MgProtoMe) in a range of mutants and conditions in which the steady-state levels of MgProto (MgProtoMe) have been modified. Overall we found that there was no correlation between the steady-state levels of MgProto (MgProtoMe) and Lhcb1 expression or with any of the other genes tested. Taking these results together, we propose that the current model on plastid signaling must be revised.

In plants, chlorophylls (chlorophyll a and chlorophyll b) are the most abundant tetrapyrrole molecules and are essential for photosynthesis. The first committed step of chlorophyll biosynthesis is the insertion of Mg(2+) into protoporphyrin IX, and thus subsequent steps of the biosynthesis are called the Mg branch. As the Mg branch in higher plants is complex, it was not until the last decade--after many years of intensive research--that most of the genes encoding the enzymes for the pathway were identified. Biochemical and molecular genetic analyses have certainly modified the classic metabolic map of tetrapyrrole biosynthesis, and only recently have the molecular mechanisms of regulatory pathways governing chlorophyll metabolism been elucidated. As a result, novel functions of tetrapyrroles and biosynthetic enzymes have been proposed. In this review, I summarize the recent findings on enzymes involved in the Mg branch, mainly in higher plants.

Insertion of magnesium into protoporphyrin IX by magnesium chelatase is a key step in the chlorophyll biosynthetic pathway, which takes place in plant chloroplasts. ATP hydrolysis by the CHLI subunit of magnesium chelatase is an essential component of this reaction, and the activity of this enzyme is a primary determinant of the rate of magnesium insertion into the chlorophyll molecule (tetrapyrrole ring). Higher plant CHLI contains highly conserved cysteine residues and was recently identified as a candidate protein in a proteomic screen of thioredoxin target proteins (Balmer, Y., Koller, A., del Val, G., Manieri, W., Schurmann, P., and Buchanan, B. B. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 370–375). To study the thioredoxin-dependent regulation of magnesium chelatase, we first investigated the effect of thioredoxin on the ATPase activity of CHLI1, a major isoform of CHLI in Arabidopsis thaliana. The ATPase activity of recombinant CHLI1 was found to be fully inactivated by oxidation and easily recovered by thioredoxin-assisted reduction, suggesting that CHLI1 is a target protein of thioredoxin. Moreover, we identified one crucial disulfide bond located in the C-terminal helical domain of CHLI1 protein, which may regulate the binding of the nucleotide to the N-terminal catalytic domain. The redox state of CHLI was also found to alter in a light-dependent manner in vivo. Moreover, we successfully observed stimulation of the magnesium chelatase activity in isolated chloroplasts by reduction. Our findings strongly suggest that chlorophyll biosynthesis is subject to chloroplast biogenesis regulation networks to coordinate them with the photosynthetic pathways in chloroplasts.

Cytokinins and light activate the transcription of the cucumber NADPH-protochlorophyllide reductase (POR) gene. We have previously reported that 2.3 kb of the 5'-region of this gene contains a cis-element that is responsive to cytokinin. In this study, to identify the cytokinin-responsive cis-element corresponding to chlorophyll biosynthesis and chloroplast development, we performed transient expression assays in etiolated cucumber cotyledons. A 5'-deletional analysis indicated that a 411-bp fragment (-451 to -40 bp) contained at least one of the cis-elements related to cytokinin-responsiveness. Gel mobility shift assays also detected cytokinin-enhanced binding in this region. DNase I footprinting analysis, using a 150-bp fragment (-490 to -340 bp) as the probe, identified the cytokinin-enhanced protected sequence as 5'-ATATTAGTGATAT-3'. More detailed gel mobility shift and competition analyses identified 5'-TATTAG-3' as the sequence critical for cytokinin-enhanced binding. Mutations in the identified sequence in the transient expression assay caused a reduced but retained cytokinin-responsiveness, as well as low reporter activity of untreated control. These results suggest that the identified sequence is a novel cis-element exhibiting cytokinin-dependent protein binding in vitro, which may function effectively when interacting with other cytokinin-related elements. The effects of this element on the chloroplast development are discussed in relation to other cytokinin-related elements.

Summary Jasmonic acid (JA) and methyl jasmonate (MeJA), collectively termed jasmonates, are ubiquitous plant signalling compounds. Several types of stress conditions, such as wounding and pathogen infection, cause endogenous JA accumulation and the expression of jasmonate-responsive genes. Although jasmonates are important signalling components for the stress response in plants, the mechanism by which jasmonate signalling contributes to stress tolerance has not been clearly defined. A comprehensive analysis of jasmonate-regulated metabolic pathways in Arabidopsis was performed using cDNA macroarrays containing 13516 expressed sequence tags (ESTs) covering 8384 loci. The results showed that jasmonates activate the coordinated gene expression of factors involved in nine metabolic pathways belonging to two functionally related groups: (i) ascorbate and glutathione metabolic pathways, which are important in defence responses to oxidative stress, and (ii) biosynthesis of indole glucosinolate, which is a defence compound occurring in the Brassicaceae family. We confirmed that JA induces the accumulation of ascorbate, glutathione and cysteine and increases the activity of dehydroascorbate reductase, an enzyme in the ascorbate recycling pathway. These antioxidant metabolic pathways are known to be activated under oxidative stress conditions. Ozone (O3) exposure, a representative oxidative stress, is known to cause activation of antioxidant metabolism. We showed that O3 exposure caused the induction of several genes involved in antioxidant metabolism in the wild type. However, in jasmonate-deficient Arabidopsis 12-oxophytodienoate reductase 3 (opr3) mutants, the induction of antioxidant genes was abolished. Compared with the wild type, opr3 mutants were more sensitive to O3 exposure. These results suggest that the coordinated activation of the metabolic pathways mediated by jasmonates provides resistance to environmental stresses.

Jasmonic acid (JA) and methyl jasmonate (MeJA), collectively known as JAs, regulate diverse physiological processes in plants, including the response to wounding. Recent reports suggest that a cyclopentenone precursor of JA, 12-oxo-phytodienoic acid (OPDA), can also induce gene expression. However, little is known about the physiological significance of OPDA-dependent gene expression. We used microarray analysis of approximately 21,500 Arabidopsis (Arabidopsis thaliana) genes to compare responses to JA, MeJA, and OPDA treatment. Although many genes responded identically to both OPDA and JAs, we identified a set of genes (OPDA-specific response genes [ORGs]) that specifically responded to OPDA but not to JAs. ORGs primarily encoded signaling components, transcription factors, and stress response-related genes. One-half of the ORGs were induced by wounding. Analysis using mutants deficient in the biosynthesis of JAs revealed that OPDA functions as a signaling molecule in the wounding response. Unlike signaling via JAs, OPDA signaling was CORONATINE INSENSITIVE 1 independent. These results indicate that an OPDA signaling pathway functions independently of JA/MeJA signaling and is required for the wounding response in Arabidopsis.

The arc3 (accumulation and replication of chloroplast) mutant of Arabidopsis thaliana has a small number of abnormally large chloroplasts in the cell, suggesting that chloroplast division is arrested in the mutant and ARC3 has an important role in the initiation of chloroplast division. To elucidate the role of ARC3, first we identified the ARC3 gene, and determined the location of ARC3 protein during chloroplast division because the localization and spatial orientation of such division factors are vital for correct chloroplast division. Sequencing analysis showed that ARC3 was a fusion of the prokaryotic FtsZ and part of the eukaryotic phosphatidylinositol-4-phosphate 5-kinase (PIP5K) genes. The PIP5K-homologous region of ARC3 had no catalytic domain but a membrane-occupation-and-recognition-nexus (MORN) repeat motif. Immunofluorescence microscopy, Western blotting analysis and in vitro chloroplast import and protease protection assays revealed that ARC3 protein was soluble, and located on the outer surface of the chloroplast in a ring-like structure at the early stage of chloroplast division. Prokaryotes have one FtsZ as a gene for division but have no ARC3 counterparts, the chimera of FtsZ and PIP5K, suggesting that the ARC3 gene might have been generated from FtsZ as another division factor during the evolution of chloroplast by endosymbiosis.

Tetrapyrrole compounds, such as chlorophylls, hemes, and phycobilins, are synthesized in many enzymatic steps. For regulation of the tetrapyrrole metabolic pathway, it is generally considered that several specific isoforms catalyzing particular enzymatic steps control the flow of tetrapyrrole intermediates by differential regulation of gene expression depending on environmental and developmental factors. However, the coordination of such regulatory steps and orchestration of the overall tetrapyrrole metabolic pathway are still poorly understood. In this study, we developed an original mini-array system, which enables the expression profiling of each gene involved in tetrapyrrole biosynthesis simultaneously with high sensitivity. With this system, we performed a transcriptome analysis of Arabidopsis seedlings in terms of the onset of greening, endogenous rhythm, and developmental control. Data presented here clearly showed that based on their expression profiles at the onset of greening, genes involved in tetrapyrrole biosynthesis can be classified into four categories, in which genes are coordinately regulated to control the biosynthesis. Moreover, genes in the same group were similarly controlled in an endogenous rhythmic manner but also by a developmental program. The physiological significance of these gene clusters is discussed.

The distinctive features of plant organs are primarily determined by organ-specific gene expression. We analyzed the expression specificity of 8809 genes in 7 organs of Arabidopsis using a cDNA macroarray system. Using relative expression (RE) values between organs, many known and unknown genes specifically expressed in each organ were identified. We also analyzed the organ specificity of various gene groups using the GRE (group relative expression) value, the average of the REs of all genes in a group. Consequently, we found that many gene groups even ribosomal protein genes, have strong organ-specific expression. Clustering of the expression profiles revealed that the 8809 genes were classified into 9 major categories. Although 3451 genes were clustered into the largest category, which showed constitutive gene expression, 266 and 1005 genes were found to be root- and silique-specific genes, respectively. By this clustering, particular gene groups which showed multi-organ-specific expression profiles, such as bud-flower-specific, stem-silique-specific or bud-flower-root-specific profiles, could be effectively identified. From these results, major features of plant organs could be characterized by their distinct profiles of global gene expression. These data of organ-specific gene expression are available at our web site: Arabidopsis thaliana Tissue-Specific Expression Database, ATTED (http://www.atted.bio.titech.ac.jp/).

The current research investigated the regulation of monogalactosyldiacylglycerol (MGDG) biosynthesis, catalyzed by MGDG synthase (MGD) (UDP-galactose:1,2-diacylglycerol 3-beta-D-galactosyltransferase; EC 2.4.1.46), during chloroplast development in cucumbers (Cucumis sativus L. cv. Aonagajibai). In etiolated seedlings, white light induced a transient increase in MGD mRNA, followed by a subsequent increase in enzyme activity. MGDG, digalactosyldiacylglycerol (DGDG), and linolenic acid (18 : 3) of both MGDG and DGDG accumulated in a light-dependent manner. Early light-dependent induction of MGD protein was also identified in isolated chloroplasts. When cotyledons were detached from seedlings, these light-induced changes diminished. However, when a synthetic cytokinin, benzyladenine, was added to the detached cotyledons, a transient increase in MGD mRNA and a linear increase in the enzyme activity were induced even in the dark. Galactolipids subsequently accumulated to some extent and 18 : 3 content also increased. MGDG fully accumulated in detached cotyledons with co-treatment of light and a cytokinin. Red light (600 nm) and far-red light (700 nm) both induced an increase in MGD mRNA and enzyme activity but far-red light did not induce an accumulation of MGDG. These results suggest that (1). galactolipid biosynthesis is regulated by the cooperation of light and a cytokinin; (2). the accumulation of MGDG requires cytokinin in addition to light; (3). a red light (600-700 nm) dependent factor is necessary for the maximal galactolipid accumulation in addition to increase in MGD transcript and activity.

To elucidate the mechanism of irradiance-dependent adjustments in the chlorophyll antenna size of photosynthesis, we addressed the regulation of expression of genes encoding a variety of chlorophyll biosynthesis enzymes and that of the Lhcb genes in the model organism Dunaliella salina. Among the chlorophyll biosynthesis enzymes tested, only the chlorophyll a oxygenase (CAO) gene responded to changes in the level of irradiance with substantial mRNA level and kinetics of change that were similar to those of the Lhcb genes. Evidence is presented for the operation of a cytosolic signal transduction pathway for the rapid (order of minutes) regulation of both CAO and Lhcb gene expression by irradiance. Inhibitor studies and transient activation of Ca2+-dependent kinase suggested phopholipase-C activation to Ca2+ release, and activation of a specific Ca2+/CaM-dependent protein kinase in this cytosolic signal transduction pathway. The redox state of the plastoquinone pool also serves to regulate CAO and Lhcb gene expression on a slower time scale (hours) and probably serves as a plastidic-origin signal that acts coordinately with the cytosolic signal transduction pathway. It is proposed that irradiance-dependent adjustments in the chlorophyll antenna size occur by coordinate regulation of CAO and Lhcb gene expression via two distinct signal transduction pathways in photosynthetic organisms.

In higher plants, glycolipids such as monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are major components of chloroplast membranes in leaves. A recent study identified an isoform of MGDG synthase that is expressed specifically in floral organs, suggesting a novel function for glycolipids in flowers. To elucidate the localization and developmental changes of glycolipids and their biosynthetic activities in flowers, we carried out a series of analytical studies with Petunia hybrida. The results showed that the biosynthetic activities of galactolipid synthesis, particularly for DGDG, increased during flower development. Among the floral organs, the pistil had the highest galactolipid synthetic activity. Its specific activity for incorporation of UDP-galactose to yield galactolipids was estimated to be more than twice that of leaves, which are the major site of galactolipid synthesis in plant tissues. Analysis of lipid contents of pistils revealed that they contained higher amounts of galactolipids than other floral organs. Moreover, DGDG was more abundant than MGDG in both pistils and petals. These results show that DGDG is a major glycolipid in floral organs and that DGDG biosynthetic activity is highly upregulated in the pistils and petals of Petunia flowers.

Unicellular microalgae hold the promise of commercial exploitation in mass culture for hydrogen and biomass production. In any microalgal production system, the achievable photosynthetic productivity and light utilization efficiency of the algae are the single most important factors in the determination of cost. Microalgal mass cultures growing under full sunlight have a low per chlorophyll (Chl) productivity since, at high photon flux densities, the rate of photon absorption by the Chl antenna far exceeds the rate at which photons can be utilized for photosynthesis. Excess photons are dissipated as fluorescence or heat. Up to 80% of absorbed photons could thus be wasted, reducing light conversion efficiencies and cellular productivity to fairly low levels. This shortcoming could possibly be alleviated by the development of microalgal strains with a limited number of Chl molecules in the light-harvesting antenna of their photosystems, i.e., strains that have a truncated Chl antenna size . It is expected that individually, such microalgae will not be able to saturate rates of photosynthesis and, thus, will not be subject to wasteful dissipation of excitation energy. In turn, the productivity of the mass culture will be improved. The method of choice to reach the objective of a “ t runcated l ight-harvesting Chl a ntenna” size ( tla ) employed DNA insertional and chemical mutagenesis of the unicellular green algae Chlamydomonas reinhardtii and Dunaliella salina , followed by a rigorous screening protocol to identify mutants with a smaller light-harvesting Chl antenna size. Molecular and genetic analyses of isolated tla strains were performed. Biochemical and physiological analyses in terms of photosynthetic productivity and light conversion efficiencies are presented. The results show that a truncated Chl antenna size of PSII is more important than that of PSI in terms of the photosynthetic productivity of a mass culture. A list of genes that confer a “ t runcated l ight-harvesting Chl a ntenna” size to green algae is being compiled for future application in algal hydrogen and biomass production.

Ferrochelatase catalyzes the insertion of Fe2+ into protoporphyrin IX to generate protoheme. In higher plants, there is evidence for two isoforms of this enzyme that fulfill different roles. Here, we describe the isolation of a second ferrochelatase cDNA from cucumber (CsFeC2) that was less similar to a previously isolated isoform (CsFeC1) than it was to some ferrochelatases from other higher plants. Inin vitro import experiments, the two cucumber isoforms showed characteristics similar to their respective ferrochelatase counterparts of Arabidopsis thaliana. The C-terminal region of CsFeC2 but not CsFeC1 contained a conserved motif found in light-harvesting chlorophyll proteins, and CsFeC2 belonged to a phylogenetic group of plant ferrochelatases containing this conserved motif. We demonstrate that CsFeC2 was localized predominantly in thylakoid membranes as an intrinsic protein, and forming complexes probably with the C-terminal conserved motif, but a minor portion was also detected in envelope membranes. CsFeC2 mRNA was detected in all tissues and was light-responsive in cotyledons, whereasCsFeC1 mRNA was detected in nonphotosynthetic tissues and was not light-responsive. Interestingly, tissue-, light-, and cycloheximide-dependent expressions of the two isoforms of ferrochelatase were similar to those of two glutamyl-tRNA reductase isoforms involved in the early step of tetrapyrrole biosynthesis, suggesting the existence of distinctly controlled tetrapyrrole biosynthetic pathways in photosynthetic and nonphotosynthetic tissues.

To elucidate the mechanism of an irradiance-dependent adjustment in the chlorophyll (Chl) antenna size of Dunaliella salina, we investigated the regulation of expression of the Chl a oxygenase (CAO) and light-harvesting complex b (Lhcb) genes as a function of Chl availability in the photosynthetic apparatus. After a high-light to low-light shift of the cultures, levels of both CAO and Lhcb transcripts were rapidly induced by about 6-fold and reached a high steady-state level within 1.5 h of the shift. This was accompanied by repair of photodamaged photosystem II (PSII) reaction centers, accumulation of Chl a and Chl b (4:1 ratio), photosystem I (PSI), light-harvesting complex, and by enlargement of the Chl antenna size of both photosystems. In gabaculine-treated cells, induction of CAO and Lhcb transcripts was not affected despite substantial inhibition in de novo Chl biosynthesis. However, cells were able to synthesize and accumulate some Chl a and Chl b (1:1 ratio), resulting in a marked lowering of the Chl a to Chl b ratio in the presence of this inhibitor. Assembly incorporation of light-harvesting complex and a corresponding Chl antenna size increase, mostly for the existing photosystems, was noted in the presence of gabaculine. Repair of photodamaged PSII was not affected by gabaculine. However, assembly accumulation of new PSI was limited under such conditions. These results suggest a coordinate regulation of CAO and Lhcb gene transcription by irradiance, independent of Chl availability. The results are discussed in terms of different signal transduction pathways for the regulation of the photosynthetic apparatus organization by irradiance.

NADPH-protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide a in the chlorophyll biosynthetic pathway. Here, we identified two distinct POR cDNAs from tobacco. Both POR isoforms are encoded by a respective single copy gene in tobacco genome. The overall deduced amino acid sequences of two tobacco cDNAs, designated here POR1 and POR2, displayed significant identities (∼75%), but showed different patterns of light and developmental regulation. In contrast to the previously isolated POR isoforms of Arabidopsis thaliana and barley, the expression of both tobacco POR isoforms were not negatively regulated by light and persisted in matured green tissues. Furthermore, the expression of both genes appeared to be regulated by a diurnal regulation. These results show a wide variety of light- and development-dependent regulations of POR gene expression among angiosperms. Furthermore, phylogenetic analysis including tobacco revealed that POR gene family is differentially represented by angiosperms, most of which is probably caused by independent gene duplication in individual plant. Present results imply a modification of the previous concept that chlorophyll biosynthesis and chloroplast differentiation in angiosperms are ubiquitously controlled by unique functions of two POR isoforms.

The colonization of land by plants was a key event in the evolution of life. Here we report the draft genome sequence of the filamentous terrestrial alga Klebsormidium flaccidum (Division Charophyta, Order Klebsormidiales) to elucidate the early transition step from aquatic algae to land plants. Comparison of the genome sequence with that of other algae and land plants demonstrate that K. flaccidum acquired many genes specific to land plants. We demonstrate that K. flaccidum indeed produces several plant hormones and homologues of some of the signalling intermediates required for hormone actions in higher plants. The K. flaccidum genome also encodes a primitive system to protect against the harmful effects of high-intensity light. The presence of these plant-related systems in K. flaccidum suggests that, during evolution, this alga acquired the fundamental machinery required for adaptation to terrestrial environments., Plant colonization of land is an important evolutionary event. Here, the authors sequence the genome of a filamentous terrestrial alga and, through a comparative analysis with related algae and land plant species, provide insight into how aquatic algae adapted to terrestrial environments.

Two distinct cDNA clones (IPI1 and IPI2) encoding IPI were isolated from Nicotiana tabacum. In situ expression of isopentenyl diphosphate isomerase-1 (IPI1)- and IPI2-green fluorescent protein fusion constructs revealed that IPI1 and IPI2 were localized in chloroplast and cytosol, respectively. The level of IPI1 mRNA was increased under high-salt and high-light stress conditions, while that of IPI2 mRNA was increased under high-salt and cold stress conditions. Both IPI transcripts were increased in an abscisic acid-independent manner. This is the first report of a cytosolic IPI. The results indicated that two distinct IPIs were differentially induced in response to stress.

Monogalactosyldiacylglycerol (MGDG) is a major constituent of thylakoid membrane in chloroplasts. Therefore, it is considered to have an important role in the maintenance of the complicated structure of the thylakoid membrane. We have succeeded in cloning the enzyme for MGDG synthesis and overexpressed it in Escherichia coli. In this study we analyzed the morphology of the E. coli harboring the gene. The fatty acid composition of its membrane lipids did not differ between the wild type and transformant, except for the appearance of MGDG. However, transformant cells appeared to be elongated. DAPI staining revealed the entire intracellular region of filamentous cells to be stained; therefore, the elongation of the cells is probably due to a defect in cell division. Atomic force microscopy revealed that the transformant had a smooth but scratched surface. It was concluded that the excessive accumulation of a non-bilayer lipid, MGDG, interfered with the translocation of proteins across the plasma membrane, including those for cell division.

), BA remarkably increased the level of mRNA of NADPH-protochlorophyllide oxidoreductase (POR, EC1.3.1.33), which catalyzes the photoreduction of protochlorophyllide a to chlorophyllide a. In this study, we examined the regulatory mechanism of the BA–induced expression of the POR gene in more detail. In etiolated cotyledons of cucumber, the levels of POR mRNA and its protein were remarkably increased by BA treatment dose- and time-dependently. The increase was more marked in the mRNA than the protein. The BA-induced increase in the mRNA was repressed by treatment with cycloheximide. A run–on assay showed that BA increases the POR mRNA at least at the transcriptional level. Moreover, a transient reporter assay of the 5'- upstream region of the POR gene reveled this region to contain a cis–acting element(s) for cytokinin response. These results indicated that the BA–induced increase in the mRNA is mainly dependent on transcriptional activation, which accompanies de novo protein synthesis in cytoplasm.

In Arabidopsis thaliana , we identified a novel gene of a NADPH-protochlorophyllide oxidoreductase (POR) isoform, which catalyzes the light-dependent protochlorophyllide a reduction in the chlorophyll (Chl) biosynthetic pathway. The deduced amino acid sequence of the novel POR isoform (PORC) showed significant identities (∼75%) with the previously isolated two POR isoforms of A. thaliana . Contrasting with these POR isoforms, the PORC transcript increased in etiolated seedlings by illumination, and was dominantly expressed in immature and mature tissues. The present results demonstrated that Chl biosynthesis and chloroplast biogenesis in A. thaliana are controlled by three POR isoforms, which are differentially controlled by light and development.

Ferrochelatase catalyzes the insertion of Fe2+ into protoporphyrin IX to generate protoheme. A putative mature region of a cucumber ferrochelatase cDNA (hemH) was overexpressed in Escherichia coli and purified to homogeneity (40 kDa). The optimum pH was 7.7, and the apparent K(m) values for deuteroporphyrin IX and Fe2+ were 14.4 microM and 4.7 microM, respectively. The activity of the ferrochelatase was inhibited by N-methylprotoporphyrin IX (I50 = 4 nM). Western blot analysis with a polyclonal antibody raised against the recombinant ferrochelatase showed that the antibody crossreacted with protein extracts from hypocotyls and roots of cucumber but not with that from cotyledons. The antibody did not crossreact with proteins of thylakoid membranes of chloroplasts in cucumber cotyledons, although the ferrochelatase activity was mainly associated with the thylakoid membranes. Northern blot analysis also indicated that the hemH gene was expressed mainly in hypocotyls and roots, but little in cotyledons, and the level of the hemH transcripts was not light-responsive. These results demonstrated that the cucumber hemH gene encodes a ferrochelatase which presumably functions for heme biosynthesis in non-photosynthetic tissues, such as hypocotyls and roots, and suggested the presence of other types of ferrochelatase in cucumber, one of which is located in thylakoid membranes of chloroplasts.

Photoreduction of protochlorophyllide a to chloro-phyllide a that is catalyzed by NADPH-protochlorophyl-lide oxidoreductase (NPR) is only a light-dependent step inoverall processes of biosynthesis of Chi in angiosperms.Unlike many other plants, in fully green leaves of cucum-ber the expression of only a single NPR gene of cucumberwas positively regulated by light and developmental age ofleaf, and also by diurnal and circadian rhythms. The singleNPR gene was shown to be involved in Chi synthesisthroughout of the vegetative growth of cucumber (in cot-yledons and fully green leaves).Key words: Chlorophyll synthesis — Circadian rhythm— Cucumber — Cucumis sativus — Diurnal rhythm— NADPH-protochlorophyllide oxidoreductase (EC1.3.1.33).Angiosperms germinated in darkness exhibit an etio-lated phenotype. In etiolated plants, etioplasts, a particulartype of plastids, are formed from proplastids. Etioplastscontain a large amount of protochlorophyllide a (Pchlidea) as a precursor of Chi. When etiolated angiosperms areilluminated, Pchlide a is photoreduced to chlorophyllide a(Chlide a) and etioplasts are transformed to photosynthet-ically active chloroplasts with concomitant breakdownof prolamellar bodies and formation of thylakoid mem-branes.The photoreduction of Pchlide a to Chlide a isthe only light-dependent reaction in the Chi synthesis ofangiosperms and is a key regulatory step in overall pro-cesses of greening of angiosperms. The photoreduction iscatalyzed by NADPH-protochlorophyllide oxidoreductase(NPR) (EC 1.3.1.33 (Ape) l et al. 1980). In many plants alarge amount of NPR protein and its mRNA accumulate in