Tetrapyrrole metabolism is involved in lesion formation, cell death, in the Arabidopsis lesion initiation 1 mutant

The Arabidopsis lesion initiation 1 (len1) mutant develops lesions on leaves without pathogen attack. The len1 plants display lesion formation as they grow under short-day conditions (SD), but not under long-day conditions (LD). This study was conducted to examine how lesion formation, viz., cell death, in len1 plants occurs under SD. I present genetic and physiological data to show that tetrapyrrole metobolism is necessary for lesion formation in len1 plants. Lesion formation was suppressed in the len1lin2 double mutant under SD. lesion initiation 2 (lin2) is another lesion mimic mutant with a defect in tetrapyrrole biosynthesis. Suppression of lesion formation in len1 plants was also observed when they were crossed with the mutants that had defects in other steps in tetrapyrrole metabolism. Suppression was correlated with reduced chlorophyll (Chl) levels in the double mutants. Furthermore, I found that dark-to-light transition caused a bleached phenotype in len1 plants, as in the case of antisense ACD1 (acd, accelerated cell death) plants. ACD1 encodes pheophorbide a oxygenase (PaO), which is involved in Chl catabolism in Arabidopsis. These results suggest that tetrapyrrole metabolism, especially Chl breakdown, might be involved in lesion formation in len1 plants.     

Interaction of the molecular chaperone HtpG with uroporphyrinogen decarboxylase in the cyanobacterium Synechococcus elongatus PCC 7942

Uroporphyrinogen decarboxylase (HemE) is important due to its location at the first branch-point in tetrapyrrole biosynthesis. We detected a complex formation between full-length polypeptides of HtpG and HemE by biochemical studies in vivo and in vitro. The interaction suppressed the enzyme activity, suggesting a regulatory role of HtpG in tetrapyrrole biosynthesis.     

Interaction of the Molecular Chaperone HtpG with Uroporphyrinogen Decarboxylase in the Cyanobacterium Synechococcus elongatus PCC 7942

Uroporphyrinogen decarboxylase (HemE) is important due to its location at the first branch-point in tetrapyrrole biosynthesis. We detected a complex formation between full-length polypeptides of HtpG and HemE by biochemical studies in vivo and in vitro. The interaction suppressed the enzyme activity, suggesting a regulatory role of HtpG in tetrapyrrole biosynthesis.     

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.     

Green or red: what stops the traffic in the tetrapyrrole pathway?

Regulation of tetrapyrrole biosynthesis is crucial to plant metabolism. The two pivotal control points are formation of the initial precursor, 5-aminolaevulinic acid (ALA), and the metal-ion insertion step: chelation of Fe(2+) into protoporphyrin IX leads to haem and phytochromobilin, whereas insertion of Mg(2+) is the first step to chlorophyll. Recent studies with mutants and transgenic plants have demonstrated that perturbation of the branch point affects ALA formation. Moreover, one of the signals that controls the expression of genes for nuclear-encoded chloroplast proteins has been shown to be Mg-protoporphyrin-IX. Here, we discuss the regulation of branch-point flux and the relative contributions of the haem and chlorophyll branches to the regulation of ALA synthesis and thus to flow through the tetrapyrrole pathway.     

RLIN1, encoding a putative coproporphyrinogen Ⅲ oxidase, is involved in lesion initiation in rice

Lesion mimic is necrotic lesions on plant leaf or stem in the absence of pathogenic infection, and its exact biological mechanism is varied. By a large-scale screening of our T-DNA mutant population, we identified a mutant rice lesion initiation 1 (rlin1), which was controlled by a single nuclear recessive gene. Map-based cloning revealed that RLIN1 encoded a putative coproporphyrinogen Ⅲ oxidase in tetrapyrrole biosynthesis pathway. Sequencing results showed that a G to T substitution occurred in the second exon of RLIN1 and led to a missense mutation from Asp to Tyr. Ectopic expression of RLIN1 could rescue rlin1 lesion mimic phenotype. Histochemical analysis demonstrated that lesion formation in rlin1 was light-dependent accompanied by reactive oxygen species accumulated. These results suggest that tetrapyrrole participates in lesion formation in rice.     

Biosynthesis of corriphyrins - a new branch of metabolism of tetrapyrrole compounds

Experiments were carried out to study biosynthesis of tetrapyrrole pigments--corriphyrins which are methylated reduced derivatives of uroporphyrin III. It was shown that the pattern of corriphyrin synthesis was close to that of vitamin B12. The mutant strain of propionic bacteria which was active in the vitamin B12 formation synthesized significantly more corriphyrins than the original strain. The corriphyrin synthesis was stimulated by delta-aminolevulinic acid and methionine and was inhibited by hydroxylamine and aeration. The formation of corriphyrins was repressed by vitamin B12. It is concluded that corriphyrins are precursors of vitamin B12 at the stage between uroporphyrinogene III and cobyrinic acid. The paper discusses further investigations of metabolism of methylated derivatives of uroporphyrinogene III as a method of elucidating the evolution of tetrapyrrole compounds and clarifying the processes involved in porphyrin metabolism in higher plants and animals.     

Involvement of free and enzyme-bound intermediates in the reaction mechanism catalyzed by the bovine liver immobilized porphobilinogen deaminase. Proof that they are substrates for cosynthetase in uroporphyrinogen III biosynthesis

The detection and accumulation of tetrapyrrole intermediates synthesized by the action of bovine liver porphobilinogen deaminase immobilized to Sepharose 4B is reported. Employing Sepharose-deaminase preparations, two phases in uroporphyrinogen I synthesis as a function of time were observed, suggesting the accumulation of free and enzyme-bound intermediates, the concentration and distribution of which were time dependent. The deaminase-bound intermediate behaves as a substrate in uroporphyrinogen I synthesis whereas the free intermediates produce enzyme inhibition. The tetrapyrrole intermediate bound to the Sepharose-enzyme is removed from the protein by the binding of porphobilinogen. Free as well as enzyme-bound intermediates are shown to be substrates for cosynthetase with formation of 80% uroporphyrinogen III.     

Light and oxygen regulation of the synthesis of bacteriochlorophylls a and c in Chloroflexus aurantiacus.

Control of the synthesis of bacteriochlorophylls (Bchls) a and c by light and oxygen was studied in Chloroflexus aurantiacus grown in batch or chemostat culture with serine as the growth-limiting substrate. For comparison, inhibition by gabaculine of the formation of selected tetrapyrroles was studied. The inhibitory effect of gabaculine decreased in the following order of tetrapyrrole formation: coproporphyrin greater than Bchl c greater than Bchl a. Not only did addition of 5-aminolevulinate (ALA) reverse the inhibition by gabaculine, it also caused an increase in Bchl c content when the cultures grew at high concentrations of ALA. Inhibition of Bchl a, Bchl c, and coproporphyrin formation by oxygen was similar to inhibition by gabaculine. Addition of ALA to aerated cultures led to significant accumulation of coproporphyrin. These results suggest that oxygen inhibits tetrapyrrole formation at a site before ALA formation. Control by light was studied with chemostat cultures transferred from 5 klx to 25 klx. This resulted in only a transient increase of the protein level of the culture, while specific contents of Bchls c and a and the ratio Bchl c/Bchl a decreased to lower steady states. However, the specific content of coproporphyrin increased. Addition of ALA to chemostat cultures adapted to 50 klx increased specific coproporphyrin and Bchl c contents by factors of about 20 and 4, respectively, while the specific Bchl a content was only slightly increased and protein levels were unaffected. Increasing the serine concentration caused an initial increase in the specific Bchl c content, which returned to the original value as soon as the protein content had attained its maximal level. These results suggest that light does not control ALA formation as strictly as oxygen and that competition of biomass formation and tetrapyrrole synthesis for common precursors may be influenced by light.     

A mitochondrial protein homologous to the mammalian peripheral-type benzodiazepine receptor is essential for stress adaptation in plants

The cloning of abiotic stress-inducible genes from the moss Physcomitrella patens led to the identification of the gene PpTSPO1, encoding a protein homologous to the mammalian mitochondrial peripheral-type benzodiazepine receptor and the bacterial tryptophane-rich sensory protein. This class of proteins is involved in the transport of intermediates of the tetrapyrrole biosynthesis pathway. Like the mammalian homologue, the PpTSPO1 protein is localized to mitochondria. The generation of PpTSPO1-targeted moss knock-out lines revealed an essential function of the gene in abiotic stress adaptation. Under stress conditions, the PpTSPO1 null mutants show elevated H(2)O(2) levels, enhanced lipid peroxidation and cell death, indicating an important role of PpTSPO1 in redox homeostasis. We hypothesize that PpTSPO1 acts to direct porphyrin precursors to the mitochondria for heme formation, and is involved in the removal of photoreactive tetrapyrrole intermediates.     

Post-translational control of tetrapyrrole biosynthesis in plants, algae, and cyanobacteria

The tetrapyrrole biosynthetic pathway provides the vital cofactors and pigments for photoautotrophic growth (chlorophyll), several essential redox reactions in electron transport chains (haem), N- and S-assimilation (sirohaem), and photomorphogenic processes (phytochromobilin). While the biochemistry of the pathway is well understood and almost all genes encoding enzymes of tetrapyrrole biosynthesis have been identified in plants, the post-translational control and organization of the pathway remains to be clarified. Post-translational mechanisms controlling metabolic activities are of particular interest since tetrapyrrole biosynthesis needs adaptation to environmental challenges. This review surveys post-translational mechanisms that have been reported to modulate metabolic activities and organization of the tetrapyrrole biosynthesis pathway.     

A novel Arabidopsis thaliana protein is a functional peripheral-type benzodiazepine receptor

A key element in the regulation of mammalian steroid biosynthesis is the 18 kDa peripheral-type benzodiazepine receptor (PBR), which mediates mitochondrial cholesterol import. PBR also possess an affinity to the tetrapyrrole metabolite protoporphyrin. The bacterial homolog to the mammalian PBR, the Rhodobacter TspO (CrtK) protein, was shown to be involved in the bacterial tetrapyrrole metabolism. Looking for a similar mitochondrial import mechanism in plants, protein sequences from Arabidopsis and several other plants were found with significant similarities to the mammalian PBR and to the Rhodobacter TspO protein. A PBR-homologous Arabidopsis sequence was cloned and expressed in E. coli. The recombinant gene product showed specific high affinity benzodiazepine ligand binding. Moreover, the protein applied to E. coli protoplasts caused an equal benzodiazepine-stimulated uptake of cholesterol and protoporphyrin IX. These results suggest that the PBR like protein is involved in steroid import and is directing protoporphyrinogen IX to the mitochondrial site of protoheme formation.     

Spectroscopic evidence for a porphobilinogen deaminase-tetrapyrrole complex that is an intermediate in the biosynthesis of uroporphyrinogen III

Incubation of porphobilinogen (PBG) with PBG deaminase from Rhodopseudomonas sphaeroides in carbonate buffer (pH 9.2) to total PBG consumption resulted in low yields of uroporphyrinogen I (uro'gen I). In the reaction mixture a pyrrylmethane accumulated, which at longer incubation periods was transformed into uro'gen I. The accumulated pyrrylmethane gave an Ehrlich reaction which was different from that of a 2-(aminomethyl)dipyrrylmethane or 2-(aminomethyl)tripyrrane. It resembled that of a bilane (tetrapyrrylmethane) but was different from that of a 2-(hydroxymethyl)bilane. The 13C NMR spectra of incubations carried out with [11-13C]PBG indicated that the pyrrylmethane was a tetrapyrrole with methylene resonances at 22.35-22.50 ppm. It was loosely bound to the deaminase, and when separated from the enzyme by gel filtration or gel electrophoresis, it immediately cyclized to uro'gen I. No enzyme-bound methylene could be detected by its chemical shift, suggesting that its line width must be very broad. When uro'gen III-cosynthase was added to the deaminase-tetrapyrrole complex, uro'gen III was formed at the expense of the latter in about 75% yield. The tetrapyrrole could only be partially displaced from the enzyme by ammonium ions, although a small amount of 2-(aminomethyl)bilane was always formed together with the tetrapyrrole intermediate. A protonated uro'gen I structure for this intermediate was ruled out by incubations using [2,11-13C]PBG. Uro'gen III formation from 2-(hydroxymethyl)bilane (HMB) and from the deaminase-tetrapyrrole intermediate was compared by using deaminase-cosynthase and cosynthase from several sources. It was found that while the HMB inhibited uro'gen III formation at higher concentrations and longer incubation times, uro'gen III formation from the complex did not decrease with time.     

Tetrapyrrole regulation of nuclear gene expression

Tetrapyrroles are the structural backbone of chlorophyll and heme, and are essential for primary photochemistry, light harvesting, and electron transport. The biochemistry of their synthesis has been studied extensively, and it has been suggested that some of the tetrapyrrole biochemical intermediates can affect nuclear gene expression. In this review, tetrapyrrole biosynthesis, which occurs in the chloroplast, and its regulation will be covered. An analysis of the intracellular location of tetrapyrrole intermediates will also be included. The focus will be on tetrapyrrole intermediates that have been suggested to affect gene expression. These include Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester. Recent evidence also suggests a specific signaling role for the H subunit of Mg-chelatase, an enzyme that catalyzes the insertion of Mg into the tetrapyrrole ring. Since gene expression studies have been done in plants and green algae, our discussion will be limited to these organisms. This revised version was published online in June 2006 with corrections to the Cover Date.     

Probing the active site of Pseudomonas aeruginosa porphobilinogen synthase using newly developed inhibitors

Porphobilinogen synthase catalyzes the first committed step of the tetrapyrrole biosynthesis pathway. In an aldol-like condensation, two molecules of 5-aminolevulinic acid (ALA) form the first pyrrole, porphobilinogen. Newly synthesized analogues of a reaction intermediate of porphobilinogen synthase have been employed in studying the active site and the catalytic mechanism of this early enzyme of tetrapyrrole biosynthesis. This study combines structural and kinetic evaluation of the inhibition potency of these inhibitors. In addition, one of the determined protein structures provides for the first time structural evidence of a magnesium ion in the active site. From these results, we can corroborate an earlier postulated enzymatic mechanism that starts with formation of a C-C bond, linking C3 of the A-side ALA to C4 of the P-side ALA through an aldole addition. The obtained data are discussed with respect to the current literature.     

Toxic tetrapyrrole accumulation in protoporphyrinogen IX oxidase-overexpressing transgenic rice plants

We generated transgenic rice plants (Oryza sativa cv. Dongjin) over-expressing human protoporphyrinogen IX oxidase (PPO) with the aim to increase mitochondrial PPO activity and confer herbicide resistance (Lee et al., Pestic Biochem Physiol 80:65-74, 2004). The transgenic plants showed during further leaf development the formation of severe necrotic spots and growth retardation. Several experiments were performed to examine the reasons for the formation of necrotic leaf lesions. Human PPO is normally located in mitochondria. An in vitro organellar import experiment revealed translocation of human PPO into pea chloroplasts, but not into mitochondria. Using a specific antibody raised against human PPO confirmed its plastidic localisation. The heme and chlorophyll contents were lower in necrotic leaves than wild-type leaves. Interestingly, mature and necrotic leaves of 12-week-old transgenic plants contained up to 14- and 24-fold more protoporphyrin IX, respectively, than mature wild-type leaves. Enhanced levels of Mg-Protoporphyrin IX, Mg-Protoporphyrin IX monomethyl ester and protochlorophyllide were concurrently observed in transgenic plants relative to wild type. Accumulated porphyrins and Mg-porphyrins likely act as photosensitizers and cause high formation of the reactive oxygen species. These high levels of tetrapyrrole intermediates correlated with increased rates of 5-aminolevulinic acid synthesis in transgenic plants. Tetrapyrrole-induced photooxidation was confirmed by increased lipid peroxidation and subsequent cell death. The transgenic phenotype is the consequence of a highly modified tetrapyrrole metabolism due to additional expression of human PPO. A possible regulatory role of PPO in graminaceous seedlings is discussed.     

Identification and expression of soul/p22HBP genes in zebrafish

The SOUL/p22HBP family is an evolutionarily ancient group of heme binding proteins with a main function as cytosolic buffer against tetrapyrrole accumulation. Structural and biochemical evidence suggest specialized roles in blood formation, necrotic cell death and chemotaxis. To date, nothing is known about the precise activity and expression patterns of this class of heme binding proteins during development. The zebrafish genome possesses five soul genes belonging to two subgroups, and no p22HBP orthologous gene. Here, spatial and temporal expression patterns are reported for zebrafish soul1, soul2 and soul4 genes. All three soul genes are maternally transcribed, and their zygotic expression takes place in unique (heart, pharynx, yolk syncytial layer, brain, eyes, lateral line) and overlapping (pronephros, pituitary gland, olfactory and otic vesicle) regions of the zebrafish embryo. Our study constitutes the first detailed analysis of soul gene expression in metazoan development, and provides the basis to understand the genetics of tetrapyrrole metabolism in a wide range of embryonic processes.     

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