Decreased and increased expression of the subunit CHL I diminishes Mg chelatase activity and reduces chlorophyll synthesis in transgenic tobacco plants

The chelation of Fe2+ and Mg2+ ions forms protoheme IX and Mg-protoporphyrin IX, respectively, and the latter is an intermediate in chlorophyll synthesis. Active magnesium protoporphyrin IX chelatase (Mg-chelatase) is an enzyme complex consisting of three different subunits. To investigate the function of the CHL I subunit of Mg-chelatase and the effects of modified Mg-chelatase activity on the tetrapyrrole biosynthetic pathway, we characterized N. tabacum transformants carrying gene constructs with the Chl I cDNA sequence in antisense and sense orientation under the control of the CaMV 35S promoter. Both elevated and diminished levels of Chl I mRNA and Chl I protein led to reduced Mg-chelatase activities, reflecting a perturbation of the assembly of the enzyme complex. The transformed plants did not accumulate the substrate of Mg-chelatase, protoporphyrin IX, but the leaves contained less chlorophyll and possessed increased chlorophyll a/b ratios, as well as a deficiency of light-harvesting chlorophyll binding proteins of photosystems I and II. The expression and activity of several tetrapyrrolic enzymes were reduced in parallel to lower the Mg-chelatase activity. Consistent with the lower chlorophyll contents, the rate-limiting synthesis of 5-aminolevulinate was also decreased in the transgenic lines analyzed. The consequence of reduced Mg-chelatase on early and late steps of chlorophyll synthesis, and on the organization of light harvesting complexes is discussed.     

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.     

Recent overview of the Mg branch of the tetrapyrrole biosynthesis leading to chlorophylls

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 Mg²⁺ 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.     

Heme biosynthesis and its regulation: towards understanding and improvement of heme biosynthesis in filamentous fungi

Heme biosynthesis in fungal host strains has acquired considerable interest in relation to the production of secreted heme-containing peroxidases. Class II peroxidase enzymes have been suggested as eco-friendly replacements of polluting chemical processes in industry. These peroxidases are naturally produced in small amounts by basidiomycetes. Filamentous fungi like Aspergillus sp. are considered as suitable hosts for protein production due to their high capacity of protein secretion. For the purpose of peroxidase production, heme is considered a putative limiting factor. However, heme addition is not appropriate in large-scale production processes due to its high hydrophobicity and cost price. The preferred situation in order to overcome the limiting effect of heme would be to increase intracellular heme levels. This requires a thorough insight into the biosynthetic pathway and its regulation. In this review, the heme biosynthetic pathway is discussed with regards to synthesis, regulation, and transport. Although the heme biosynthetic pathway is a highly conserved and tightly regulated pathway, the mode of regulation does not appear to be conserved among eukaryotes. However, common factors like feedback inhibition and regulation by heme, iron, and oxygen appear to be involved in regulation of the heme biosynthesis pathway in most organisms. Therefore, they are the initial targets to be investigated in Aspergillus niger.     

A gene cluster inChlorobium vibrioforme encoding the first enzymes of chlorophyll biosynthesis

A cloned 5.8-kb genomic fragment of the green sulfur bacteriumChlorobium vibrioforme encodes the genes for three enzymes catalyzing early steps in the biosynthetic pathway of tetrapyrroles, common to chlorophyll and heme. ThehemA, hemC andhemD genes encode the enzymes glutamyl tRNA dehydrogenase, porphobilinogen deaminase and uroporphyrinogen III synthase, respectively. The cloned genes were expressed in transformedEscherichia coli orSalmonella typhimurium and conferred autotrophy on the respective auxotrophs. Activities of the enzymes encoded by the cloned genes were demonstrated in vitro, with cell extracts obtained from the transformed enterobacteria. The proximity of these genes indicates that they form a cluster inChlorobium vibrioforme, while in most other organisms they appear to be scattered. The presence of this cluster may imply coordinate regulation of the genes involved and they may constitute an operon.     

Mechanisms of regulation and interplastid localization of chlorophyll biosynthesis.

The current concepts of chlorophyll biosynthesis, its interplastid localization, biosynthetic and biochemical heterogeneity, mechanisms of regulation of the key reactions, formation of 5-aminolevulinic acid and incorporation of magnesium into protoporphyrin IX, are reviewed. The literature and author's data demonstrate the existence of in vivo multienzyme systems synthesizing chlorophyll and its precursors as monovinyl and divinyl chemical species. Both types of the multienzyme systems synthesize 5-aminolevulinic acid and regulate this process independently. A hypothesis is considered that the function of the magnesium branch of chlorophyll biosynthesis in vivo is controlled by a mechanism through inhibition of the enzymes by their products because of the limitation of the binding sites for them in the membrane. An additional influence of light on the Mg-chelatase activity not only via the photosynthetic supply with ATP but also through the light-induced synthesis of the enzyme molecules de novo is described. Efficient energy migration from protoporphyrin IX and Mg-protoporphyrin IX (monomethyl ester) molecules to the protochlorophyllide active form detected by the author is discussed considering a close location of these pigments in plastid membranes and the enzymes participating in their formation.     

Identification of rate-limiting steps in yeast heme biosynthesis

The heme biosynthesis pathway in the yeast Saccharomyces cerevisiae is a highly regulated system, but the mechanisms accounting for this regulation remain unknown. In an attempt to identify rate-limiting steps in heme synthesis, which may constitute potential regulatory points, we constructed yeast strains overproducing two enzymes of the pathway: the porphobilinogen synthase (PBG-S) and deaminase (PBG-D). Biochemical analysis of the enzyme-overproducing strains revealed intracellular porphobilinogen and porphyrin accumulation. These results indicate that both enzymes play a rate-limiting role in yeast heme biosynthesis.     

Chloroplasts as source and target of cellular redox regulation: a discussion on chloroplast redox signals in the context of plant physiology

During the evolution of plants, chloroplasts have lost the exclusive genetic control over redox regulation and antioxidant gene expression. Together with many other genes, all genes encoding antioxidant enzymes and enzymes involved in the biosynthesis of low molecular weight antioxidants were transferred to the nucleus. On the other hand, photosynthesis bears a high risk for photo-oxidative damage. Concomitantly, an intricate network for mutual regulation by anthero- and retrograde signals has emerged to co-ordinate the activities of the different genetic and metabolic compartments. A major focus of recent research in chloroplast regulation addressed the mechanisms of redox sensing and signal transmission, the identification of regulatory targets, and the understanding of adaptation mechanisms. In addition to redox signals communicated through signalling cascades also used in pathogen and wounding responses, specific chloroplast signals control nuclear gene expression. Signalling pathways are triggered by the redox state of the plastoquinone pool, the thioredoxin system, and the acceptor availability at photosystem I, in addition to control by oxolipins, tetrapyrroles, carbohydrates, and abscisic acid. The signalling function is discussed in the context of regulatory circuitries that control the expression of antioxidant enzymes and redox modulators, demonstrating the principal role of chloroplasts as the source and target of redox regulation.     

Tetrapyrrole synthesis of photosynthetic chromerids is likely homologous to the unusual pathway of apicomplexan parasites

Most photosynthetic eukaryotes synthesize both heme and chlorophyll via a common tetrapyrrole biosynthetic pathway starting from glutamate. This pathway was derived mainly from cyanobacterial predecessor of the plastid and differs from the heme synthesis of the plastid-lacking eukaryotes. Here, we show that the coral-associated alveolate Chromera velia, the closest known photosynthetic relative to Apicomplexa, possesses a tetrapyrrole pathway that is homologous to the unusual pathway of apicomplexan parasites. We also demonstrate that, unlike other eukaryotic phototrophs, Chromera synthesizes chlorophyll from glycine and succinyl-CoA rather than glutamate. Our data shed light on the evolution of the heme biosynthesis in parasitic Apicomplexa and photosynthesis-related biochemical processes in their ancestors.     

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.     

Novel inhibitors of glutamyl-tRNA(Glu) reductase identified through cell-based screening of the heme/chlorophyll biosynthetic pathway

The metabolite 5-aminolevulinic acid (ALA) is an early committed intermediate in the biosynthetic pathway of heme and chlorophyll formation. In plants, 5-aminolevulinic acid is synthesized via a two-step pathway in which glutamyl-tRNA(Glu) is reduced by glutamyl-tRNA(Glu) reductase (GluTR) to glutamate 1-semialdehyde, followed by transformation to 5-aminolevulinic acid catalyzed by glutamate 1-semialdehyde aminotransferase. Using an Escherichia coli cell-based high-throughput assay to screen small molecule libraries, we identified several chemical classes that specifically inhibit heme/chlorophyll biosynthesis at this point by demonstrating that the observed cell growth inhibition is reversed by supplementing the medium with 5-aminolevulinic acid. These compounds were further tested in vitro for inhibition of the purified enzymes GluTR and glutamate 1-semialdehyde aminotransferase as confirmation of the specificity and site of action. Several promising compounds were identified from the high-throughput screen that inhibit GluTR with an I(0.5) of less than 10 microM. Our results demonstrate the efficacy of cell-based high-throughput screening for identifying inhibitors of 5-aminolevulinic acid biosynthesis, thus representing the first report of exogenous inhibitors of this enzyme.