Separate physiological roles and subcellular compartments for two tetrapyrrole biosynthetic pathways in Euglena gracilis

delta-Aminolevulinic acid (ALA), the first committed precursor to the tetrapyrrole components of hemes and chlorophylls, is synthesized by two different routes in the photosynthetic phytoflagellate Euglena gracilis: directly from glutamate, mediated by a 5-carbon pathway, and via condensation of glycine and succinyl-CoA, catalyzed by the enzyme ALA synthase. The physiological roles of the two pathways were determined by administration of specifically 14C-labeled ALA precursors to cultures growing under different physiological conditions. Relative activities of the ALA synthase and 5-carbon pathways were monitored by incorporation of radioactivity from [2-14C] glycine and [1-14C]glutamate into highly purified protoheme, heme a and chlorophyll a derivatives. Wild type cells grown photoautotrophically or photoheterotrophically synthesized chlorophyll and incorporated radioactivity from [1-14C]glutamate into the tetrapyrrole nucleus of the pigment. [2-14C]Glycine was incorporated primarily into the nontetrapyrrole-derived portions of chlorophyll. In the same cultures both [2-14C]glycine and [1-14C]glutamate were efficiently incorporated into protoheme, while only [2-14C] glycine was incorporated into heme a. In dark-grown wild type or light-grown aplastidic cells, no chlorophyll was formed, and both protoheme and heme a were labeled exclusively from [2-14C]glycine. These results indicate: (a) ALA synthase and the 5-carbon pathway operate simultaneously in growing green cells; (b) the 5-carbon pathway provides ALA for chloroplast protoheme and chlorophyll, and is associated with chloroplast development; (c) ALA synthase provides ALA only for nonplastid heme biosynthesis; and (d) the two ALA pathways are separately compartmentalized along with complete sets of enzymes for subsequent tetrapyrrole synthesis from each ALA pool. The protoheme that was synthesized from [1-14C] glutamate had a higher specific radioactivity than chlorophyll synthesized from the same precursor. This result together with calculated specific radioactivities of the products synthesized during the incubation period, suggest that both protoheme and heme a undergo metabolic turnover.     

Regulation of the tetrapyrrole biosynthetic pathway leading to heme and chlorophyll in plants and cyanobacteria

Photosynthetic organisms synthesize chlorophylls, hemes, and bilin pigments via a common tetrapyrrole biosynthetic pathway. This review summarizes current knowledge about the regulation of this pathway in plants, algae, and cyanobacteria. Particular emphasis is placed on the regulation of glutamate-1-semialdehyde formation and on the channelling of protoporphyrin IX into the heme and chlorophyll branches. The potential role of chlorophyll molecules that are not bound to photosynthetic pigment-protein complexes ('free chlorophylls') or of other Mg-containing porphyrins in regulation of tetrapyrrole synthesis is also discussed.     

Identification of plant-like galactolipids in Chromera velia, a photosynthetic relative of malaria parasites

Apicomplexa are protist parasites that include Plasmodium spp., the causative agents of malaria, and Toxoplasma gondii, responsible for toxoplasmosis. Most Apicomplexa possess a relict plastid, the apicoplast, which was acquired by secondary endosymbiosis of a red alga. Despite being nonphotosynthetic, the apicoplast is otherwise metabolically similar to algal and plant plastids and is essential for parasite survival. Previous studies of Toxoplasma gondii identified membrane lipids with some structural features of plastid galactolipids, the major plastid lipid class. However, direct evidence for the plant-like enzymes responsible for galactolipid synthesis in Apicomplexan parasites has not been obtained. Chromera velia is an Apicomplexan relative recently discovered in Australian corals. C. velia retains a photosynthetic plastid, providing a unique model to study the evolution of the apicoplast. Here, we report the unambiguous presence of plant-like monogalactosyldiacylglycerol and digalactosyldiacylglycerol in C. velia and localize digalactosyldiacylglycerol to the plastid. We also provide evidence for a plant-like biosynthesis pathway and identify candidate galactosyltranferases responsible for galactolipid synthesis. Our study provides new insights in the evolution of these important enzymes in plastid-containing eukaryotes and will help reconstruct the evolution of glycerolipid metabolism in important parasites such as Plasmodium and Toxoplasma.     

Cytokinin effects on tetrapyrrole biosynthesis and photosynthetic activity in barley seedlings

Cytokinin promotes morphological and physiological processes including the tetrapyrrole biosynthetic pathway during plant development. Only a few steps of chlorophyll (Chl) biosynthesis, exerting the phytohormonal influence, have been individually examined. We performed a comprehensive survey of cytokinin action on the regulation of tetrapyrrole biosynthesis with etiolated and greening barley seedlings. Protein contents, enzyme activities and tetrapyrrole metabolites were analyzed for highly regulated metabolic steps including those of 5-aminolevulinic acid (ALA) biosynthesis and enzymes at the branch point for protoporphyrin IX distribution to Chl and heme. Although levels of the two enzymes of ALA synthesis, glutamyl-tRNA reductase and glutamate 1-semialdehyde aminotransferase, were elevated in dark grown kinetin-treated barley seedlings, the ALA synthesis rate was only significantly enhanced when plant were exposed to light. While cytokinin do not stimulatorily affect Fe-chelatase activity and heme content, it promotes activities of the first enzymes in the Mg branch, Mg protoporphyrin IX chelatase and Mg protoporphyrin IX methyltransferase, in etiolated seedlings up to the first 5 h of light exposure in comparison to control. This elevated activities result in stimulated Chl biosynthesis, which again parallels with enhanced photosynthetic activities indicated by the photosynthetic parameters F _V/F _M, J _CO2max and J _CO2 in the kinetin-treated greening seedlings during the first hours of illumination. Thus, cytokinin-driven acceleration of the tetrapyrrole metabolism supports functioning and assembly of the photosynthetic complexes in developing chloroplasts.     

Redox regulation of chlorophyll biosynthesis

Chlorophyll captures and redirects light-energy and is thus essential for photosynthetic organisms. The demand for chlorophyll differs throughout the day and night and in response to changing light conditions. Moreover, the chlorophyll biosynthesis pathway is up to certain points shared between the different tetrapyrroles; chlorophyll, heme, siroheme and phytochromobilin, for which the cell has different requirements at different time points. Combined with the phototoxic properties of tetrapyrroles which, if not properly protected, can lead to formation of reactive oxygen species (ROS), the need for a strict regulation of the chlorophyll biosynthetic pathway is obvious. Here we describe the current knowledge on regulation of chlorophyll biosynthesis in plants by the chloroplast redox state with emphasis on the Mg-chelatase situated at the branch point between the heme and the chlorophyll pathway. We discuss the proposed role of the Mg-chelatase as a key regulator of the tetrapyrrole pathway by its effect on enzymes both up- and downstream in the pathway and we specifically describe how redox state might regulate the Mg-branch. Finally, we propose that a recently identified NADPH-dependent thioredoxin reductase (NTRC) could be involved in redox regulation or protection of chlorophyll biosynthetic enzymes and describe the possible modes of action by this enzyme.     

Evolution of the apicoplast and its hosts: from heterotrophy to autotrophy and back again

The photosynthetic origin of apicomplexan parasites was proposed upon the discovery of a reduced non-photosynthetic plastid termed the apicoplast in their cells. Although it is clear that the apicoplast has evolved through a secondary endosymbiosis, its particular origin within the red or green plastid lineage remains controversial. The recent discovery of Chromera velia, the closest known photosynthetic relative to apicomplexan parasites, sheds new light on the evolutionary history of alveolate plastids. Here we review our knowledge on the evolutionary history of Apicomplexa and particularly their plastids, with a focus on the pathway by which they evolved from free-living heterotrophs through photoautotrophs to omnipresent obligatory intracellular parasites. New sequences from C. velia (histones H2A, H2B; GAPDH, TufA) and phylogenetic analyses are also presented and discussed here.     

Yeast 5-aminolevulinate synthase provides additional chlorophyll precursor in transgenic tobacco

Synthesis of the tetrapyrrole precursor 5-aminolevulinate (ALA) in plants starts with glutamate and is a tRNA-dependent pathway consisting of three enzymatic steps localized in plastids. In animals and yeast, ALA is formed in a single step from succinyl CoA and glycine by aminolevulinate synthase (ALA-S) in mitochondria. A gene encoding a fusion protein of yeast ALA-S with an amino-terminal transit sequence for the small subunit of ribulose bisphosphate carboxylase was introduced into the genome of wild-type tobacco and a chlorophyll-deficient transgenic line expressing glutamate 1-semi-aldehyde aminotransferase (GSA-AT) antisense RNA. Expression of ALA-S in the GSA-AT antisense transgenic line provided green-pigmented co-transformants similar to wild-type in chlorophyll content, while transformants derived from wild-type plants did not show phenotypical changes. The capacity to synthesize ALA and chlorophyll was increased in transformed plants, indicating a contribution of ALA-S to the ALA supply for chlorophyll synthesis. ALA-S activity was detected in plastids of the transformants. Preliminary evidence is presented that succinyl CoA, the substrate for ALA-S, can be synthesized and metabolized in plastids. The transgenic plants formed chlorophyll in the presence of gabaculine, an inhibitor of GSA-AT. Steady-state RNA and protein levels and, consequently, the enzyme activity of GSA-AT were reduced in plants expressing ALA-S. In analogy to the light-dependent ALA synthesis attributed to feedback regulation, a mechanism at the level of intermediates or tetrapyrrole end-products is proposed, which co-ordinates the need for heme and chlorophyll precursors and restricts synthesis of ALA by regulating GSA-AT gene expression. The genetically engineered tobacco plants containing the yeast ALA-S activity demonstrate functional complementation of the catalytic activity of the plant ALA-synthesizing pathway and open strategies for producing tolerance against inhibitors of the C5 pathway.     

Fe- but not Mg-protophorphyrin IX binds to a transmembrane b-type cytochrome

Transmembrane b-type cytochromes, which are crucially involved in electron transfer chains, bind one or more heme (Fe-protoporphyrin IX) molecules non-covalently. Similarly, chlorophylls are typically also non-covalently bound by several membrane integral polypeptides involved in photosynthesis. While both, chlorophyll and heme, are tetrapyrrole macrocycles, they have different substituents at the tetrapyrrole ring moiety. Furthermore, the central metal ion is Mg²⁺ in chlorophyll and Fe^2+/3+ in heme. As heme and chlorophyll a have similar structures and might both be ligated by two histidine residues of a polypeptide chain, and as the local concentration of chlorophyll a might be up to 100-times higher than the concentration of heme, the question arises, as to how an organism ensures specific binding of heme, but not of chlorophyll, to transmembrane apo-cytochromes involved in photosynthetic electron transfer reactions. As shown here, Fe-protoporphyrin IX derivatives with modified substituents at the tetrapyrrole ring moiety still bind to an apo-cytochrome; however, association appears to be reduced. This indicates that hydrophobic and polar interactions of the ring substituents with the protein moiety stabilize the protein/heme-complex but are not essential per se. However, removal or replacement of the central Fe-ion completely abolishes formation of a holo-protein complex, and thus the central iron ion appears to determine heme binding to apo-cytochrome b₆.     

Tetrapyrrole‐based drought stress signalling

Tetrapyrroles such as chlorophyll and heme play a vital role in primary plant metabolic processes such as photosynthesis and respiration. Over the past decades, extensive genetic and molecular analyses have provided valuable insights into the complex regulatory network of the tetrapyrrole biosynthesis. However, tetrapyrroles are also implicated in abiotic stress tolerance, although the mechanisms are largely unknown. With recent reports demonstrating that modified tetrapyrrole biosynthesis in plants confers wilting avoidance, a component physiological trait to drought tolerance, it is now timely that this pathway be reviewed in the context of drought stress signalling. In this review, the significance of tetrapyrrole biosynthesis under drought stress is addressed, with particular emphasis on the inter‐relationships with major stress signalling cascades driven by reactive oxygen species (ROS) and organellar retrograde signalling. We propose that unlike the chlorophyll branch, the heme branch of the pathway plays a key role in mediating intracellular drought stress signalling and stimulating ROS detoxification under drought stress. Determining how the tetrapyrrole biosynthetic pathway is involved in stress signalling provides an opportunity to identify gene targets for engineering drought‐tolerant crops.     

FLU, a negative feedback regulator of tetrapyrrole biosynthesis, is physically linked to the final steps of the Mg(++)-branch of this pathway

Regulation of tetrapyrrole biosynthesis in higher plants has been attributed to negative feedback control. Two effectors of feedback inhibition have been identified, heme and the FLU protein. Inhibition by heme implicates the Fe-branch via regulation of the initial step of tetrapyrrole synthesis. In the present work a FLU-containing chloroplast membrane complex was identified, that besides FLU comprises the four enzymes catalyzing the final steps of chlorophyll synthesis. The results support the notion that FLU links chlorophyll synthesis and the target of feedback control, glutamyl-tRNA reductase, thereby allowing also the Mg-branch to control the initial step of tetrapyrrole synthesis.     

Multiple deletions of small Cab-like proteins in the cyanobacterium Synechocystis sp. PCC 6803: consequences for pigment biosynthesis and accumulation

Deletion of the genes for four or five small Cab-like proteins (SCPs) in photosystem (PS) I-less and PS I-less/PS II-less strains of Synechocystis sp. PCC 6803 caused a large decrease in the chlorophyll and carotenoid content of the cells without accumulation of early intermediates in the chlorophyll biosynthesis pathway, suggesting limited chlorophyll availability. The PS II/PS I ratio increased upon deletion of multiple SCPs in a wild type background, similar to what is observed in the presence of subsaturating concentrations of gabaculin, an inhibitor of an early step in the tetrapyrrole biosynthesis pathway. Upon deletion of multiple SCPs, neither 77 K fluorescence emission properties of phycobilisomeless thylakoids from the PS I-less/PS II-less strain nor the energy trapping efficiency of PS II were affected, indicating that under steady-state conditions SCPs do not bind much chlorophyll and do not serve as PS II antenna. Under conditions where protochlorophyllide reduction and thus chlorophyll synthesis were inhibited, chlorophyll disappeared quickly in a mutant lacking all five SCPs. This implies a role of SCPs in stabilization of chlorophyll-binding proteins and/or in reuse of chlorophylls. Under these conditions of inhibited reduction of protochlorophyllide, the accumulation kinetics of this intermediate were greatly altered in the absence of the five SCPs. This indicates an alteration of tetrapyrrole biosynthesis kinetics by SCPs. Based on this and other evidence, we propose that SCPs bind carotenoids and transiently bind chlorophyll, aiding in the supply of chlorophyll to nascent or reassembling photosynthetic complexes, and regulate the tetrapyrrole biosynthesis pathway as a function of the demand for chlorophyll.     

Enzymes of the heme biosynthetic pathway in the nonphotosynthetic alga Polytomella sp

Heme biosynthesis involves a number of enzymatic steps which in eukaryotes take place in different cell compartments. Enzyme compartmentalization differs between photosynthetic and nonphotosynthetic eukaryotes. Here we investigated the structures and subcellular localizations of three enzymes involved in the heme pathway in Polytomella sp., a colorless alga evolutionarily related to the green alga Chlamydomonas reinhardtii. Functional complementation of Escherichia coli mutant strains was used to isolate cDNAs encoding three heme biosynthetic enzymes, glutamate-1-semialdehyde aminotransferase, protoporphyrinogen IX oxidase, and ferrochelatase. All three proteins show highest similarity to their counterparts in photosynthetic organisms, including C. reinhardtii. All three proteins have N-terminal extensions suggestive of intracellular targeting, and immunoblot studies indicate their enrichment in a dense cell fraction that is enriched in amyloplasts. These results suggest that even though the plastids of Polytomella sp. are not photosynthetically active, they are the major site of heme biosynthesis. The presence of a gene for glutamate-1-semialdehyde aminotransferase suggests that Polytomella sp. uses the five-carbon pathway for synthesis of the heme precursor 5-aminolevulinic acid.     

Origin and evolution of the light-dependent protochlorophyllide oxidoreductase (LPOR) genes

Abstract: Light‐dependent NADPH‐protochlorophyllide oxidoreductase (LPOR) is a nuclear‐encoded chloroplast protein in green algae and higher plants which catalyzes the light‐dependent reduction of protochlorophyllide to chlorophyllide. Light‐dependent chlorophyll biosynthesis occurs in all oxygenic photosynthetic organisms. With the exception of angiosperms, this pathway coexists with a separate light‐independent chlorophyll biosynthetic pathway, which is catalyzed by light‐independent protochlorophyllide reductase (DPOR) in the dark. In contrast, the light‐dependent function of chlorophyll biosynthesis is absent from anoxygenic photosynthetic bacteria. Consequently, the question is whether cyanobacteria are the ancestors of all organisms that conduct light‐dependent chlorophyll biosynthesis. If so, how did photosynthetic eukaryotes acquire the homologous genes of LPOR in their nuclear genomes? The large number of complete genome sequences now available allow us to detect the evolutionary history of LPOR genes by conducting a genome‐wide sequence comparison and phylogenetic analysis. Here, we show the results of a detailed phylogenetic analysis of LPOR and other functionally related enzymes in the short chain dehydrogenase/reductase (SDR) family. We propose that the LPOR gene originated in the cyanobacterial genome before the divergence of eukaryotic photosynthetic organisms. We postulated that the photosynthetic eukaryotes obtained their LPOR homologues through endosymbiotic gene transfer.     

Metabolic control of the tetrapyrrole biosynthetic pathway for porphyrin distribution in the barley mutant albostrians

The barley line albostrians exhibits a severe block in chloroplast development as a result of a mutationally induced lack of plastid ribosomes. White leaves of this mutant contain undifferentiated plastids, possess only traces of chlorophyll (Chl), and are photosynthetically inactive. Chl deficiency, combined with a continuous heme requirement, should lead to drastic changes in the tetrapyrrole metabolism in white versus green leaves. We analyzed the extent to which the synthesis rate of the pathway and the porphyrin distribution toward the Chl- and heme-synthesizing bifurcation is altered in the white tissue of albostrians. Expression and activity of several distinctively regulated enzymes, such as glutamyl-tRNAglu reductase, glutamate 1-semialdehyde aminotransferase, Mg- and Fe-chelatase, and Chl synthetase, were altered in white mutant leaves in comparison to control leaves. A drastic loss in the rate-limiting formation of 5-aminolevulinate and in the Mg-chelatase and Mg-protoporphyrin IX methyltransferase activity, as well as an increase in Fe-chelatase activity, accounts for a decrease in the metabolic flux and the re-direction of metabolites. It is proposed that the tightly balanced control of activities in the pathway functions by different metabolic feedback loops and in response to developmental state and physiological requirements. This data supports the idea that the initial steps of Mg-porphyrin synthesis contribute to plastid-derived signaling toward the nucleus. The barley mutant albostrians proved to be a valuable system for studying regulation of tetrapyrrole biosynthesis and their involvement in the bi-directional communication between plastids and nucleus.