Protoporphyrin Monomethyl Ester Research Articles

A novel tetratricopeptide-repeat protein, TTP1, forms complexes with glutamyl-tRNA reductase and protochlorophyllide oxidoreductase during tetrapyrrole biosynthesis.

The biosynthesis of the tetrapyrrole end-products chlorophyll and heme depends on a multifaceted control mechanism that acts primarily at the post-translational level upon the rate-limiting step of 5-aminolevulinic acid synthesis and upon light-dependent protochlorophyllide oxidoreductase (POR). These regulatory processes require auxiliary factors that modulate the activity, stability, complex formation, and subplastidal localization of the relevant proteins. Together, they ensure optimal metabolic flow during the day and at night. As an Arabidopsis homolog of the POR-interacting tetratricopeptide-repeat protein (Pitt) first reported in Synechocystis, we characterize tetrapyrrole biosynthesis-regulating tetratricopeptide-repeat protein1 (TTP1). TTP1 is a plastid-localized, membrane-bound factor that interacts with POR, the Mg protoporphyrin monomethylester cyclase CHL27, glutamyl-tRNA reductase (GluTR), GluTR-binding protein, and FLUORESCENCE IN BLUE LIGHT. Lack of TTP1 leads to accumulation of GluTR, enhanced 5-aminolevulinic acid synthesis and lower levels of POR. Knockout mutants show enhanced sensitivity to reactive oxygen species and a slower greening of etiolated seedlings. Based on our studies, the interaction of TTP1 with GluTR and POR does not directly inhibit their enzymatic activity and contribute to the control of 5-aminolevulinic acid synthesis. Instead, we propose that TTP1 sequesters a fraction of these proteins on the thylakoid membrane, and contributes to their stability.

Induced Deactivation of Genes Encoding Chlorophyll Biosynthesis Enzymes Disentangles Tetrapyrrole-Mediated Retrograde Signaling

In photosynthetic organisms, tetrapyrrole-mediated retrograde signals are proposed to contribute to a balanced nuclear gene expression (NGE) in response to metabolic activity in chloroplasts. We followed an experimental short-term approach that allowed the assessment of modified NGE during the first hours of specifically modified enzymatic steps of the Mg branch of tetrapyrrole biosynthesis, when pleiotropic effects of other signals can be avoided. In response to 24-h-induced silencing of CHLH, CHLM, and CHL27 encoding the CHLH subunit of Mg chelatase, the Mg protoporphyrin methyltransferase and Mg protoporphyrin monomethylester cyclase, respectively, deactivated gene expression rapidly led to reduced activity of the corresponding enzymes and altered Mg porphyrin levels. But NGE was not substantially altered. When these three genes were continuously inactivated for up to 4 d, changes of transcript levels of nuclear genes were determined. CHL27 silencing for more than 24h results in necrotic leaf lesions and modulated transcript levels of oxidative stress-responsive and photosynthesis-associated nuclear genes (PhANGs). The prolonged deactivation of CHLH and CHLM results in slightly elevated transcript levels of PhANGs and tetrapyrrole-associated genes. These time-resolved studies indicate a complex scenario for the contribution of tetrapyrrole biosynthesis on NGE mediated by (1)O2-induced signaling and feedback-regulated ALA synthesis.

LCAA, a Novel Factor Required for Magnesium Protoporphyrin Monomethylester Cyclase Accumulation and Feedback Control of Aminolevulinic Acid Biosynthesis in Tobacco

Low Chlorophyll Accumulation A (LCAA) antisense plants were obtained from a screen for genes whose partial down-regulation results in a strong chlorophyll deficiency in tobacco (Nicotiana tabacum). The LCAA mutants are affected in a plastid-localized protein of unknown function, which is conserved in cyanobacteria and all photosynthetic eukaryotes. They suffer from drastically reduced light-harvesting complex (LHC) contents, while the accumulation of all other photosynthetic complexes per leaf area is less affected. As the disturbed accumulation of LHC proteins could be either attributable to a defect in LHC biogenesis itself or to a bottleneck in chlorophyll biosynthesis, chlorophyll synthesis rates and chlorophyll synthesis intermediates were measured. LCAA antisense plants accumulate magnesium (Mg) protoporphyrin monomethylester and contain reduced protochlorophyllide levels and a reduced content of CHL27, a subunit of the Mg protoporphyrin monomethylester cyclase. Bimolecular fluorescence complementation assays confirm a direct interaction between LCAA and CHL27. 5-Aminolevulinic acid synthesis rates are increased and correlate with an increased content of glutamyl-transfer RNA reductase. We suggest that LCAA encodes an additional subunit of the Mg protoporphyrin monomethylester cyclase, is required for the stability of CHL27, and contributes to feedback-control of 5-aminolevulinic acid biosynthesis, the rate-limiting step of chlorophyll biosynthesis.

Fe sparing and Fe recycling contribute to increased superoxide dismutase capacity in iron-starved Chlamydomonas reinhardtii.

Fe deficiency is one of several abiotic stresses that impacts plant metabolism because of the loss of function of Fe-containing enzymes in chloroplasts and mitochondria, including cytochromes, FeS proteins, and Fe superoxide dismutase (FeSOD). Two pathways increase the capacity of the Chlamydomonas reinhardtii chloroplast to detoxify superoxide during Fe limitation stress. In one pathway, MSD3 is upregulated at the transcriptional level up to 10(3)-fold in response to Fe limitation, leading to synthesis of a previously undiscovered plastid-specific MnSOD whose identity we validated immunochemically. In a second pathway, the plastid FeSOD is preferentially retained over other abundant Fe proteins, heme-containing cytochrome f, diiron magnesium protoporphyrin monomethyl ester cyclase, and Fe2S2-containing ferredoxin, demonstrating prioritized allocation of Fe within the chloroplast. Maintenance of FeSOD occurs, after an initial phase of degradation, by de novo resynthesis in the absence of extracellular Fe, suggesting the operation of salvage mechanisms for intracellular recycling and reallocation.

Comparative functional analysis of two hypothetical chloroplast open reading frames (ycf) involved in chlorophyll biosynthesis from Synechocystis sp. PCC6803 and plants

Hypothetical chloroplast open reading frames (ycfs) are highly conserved and interspecifically occurring genes in plastomes of plants and algae with significant functions in gene expression and photosynthesis. However, the function of many ycfs is still in vain so that attention is directed to other chloroplast functions such as metabolism of co-factors, protein translocation and protection against abiotic stress. We provide a comprehensive functional description of ycf53 and ycf59, two genes involved in chlorophyll biosynthesis. While ycf59 encodes an essential enzymatic component of Mg protoporphyrin monomethylester cyclase, ycf53 encodes a posttranslational regulator of chlorophyll biosynthesis. Their roles in tetrapyrrole biosynthesis were compared by using cyanobacterial and plant mutants with modulated expression of these two genes. Our work provides indications for diverse effects of these homologous gene products in plants and cyanobacteria on tetrapyrrole biosynthesis and photosynthesis.

Mg Protoporphyrin Monomethylester Cyclase Deficiency and Effects on Tetrapyrrole Metabolism in Different Light Conditions

Mg protoporphyrin monomethylester (MgProtoME) cyclase catalyzes isocyclic ring formation to form divinyl protochlorophyllide. The CHL27 protein is part of the cyclase complex. Deficiency of CHL27 has been previously reported to compromise photosynthesis and nuclear gene expression. In a comprehensive analysis of different CHL27 antisense tobacco lines grown under different light conditions, the physiological consequences of gradually reduced CHL27 expression on the tetrapyrrole biosynthetic pathway were explored. Excessive amounts of MgProtoME, the substrate of the cyclase reaction, accumulated in response to the reduced CHL27 content. Moreover, 5-aminolevulinic acid (ALA) synthesis, Mg chelatase and Mg protoporphyrin methyltransferase activities were reduced in transgenic plants. Compared with growth under continuous light exposure, the CHL27-deficient plants showed a stronger reduction in Chl content, cell death and leaf necrosis during diurnal light/dark cycles. This photooxidative phenotype correlated with a rapidly increasing MgProtoME steady-state level at the beginning of each light period. In contrast, the same transformants grown under continuous light exposure possessed a permanently elevated amount of MgProtoME. Its lower phototoxicity correlated with increased activities of ascorbate peroxidase and catalase, and a higher amount of reduced ascorbate. It is proposed that improved stress acclimation during continuous light in comparison with light-dark growth increases the capacity to prevent photooxidation by excess tetrapyrrole precursors and lowers the susceptibility to secondary photodynamic damage.

Differential requirement of two homologous proteins encoded by sll1214 and sll1874 for the reaction of Mg protoporphyrin monomethylester oxidative cyclase under aerobic and micro-oxic growth conditions

The two open reading frames in the Synechocystis sp. PCC 6803 genome, sll1214 and sll1874, here designated cycI and cycII, respectively, encode similar proteins, which are involved in the Mg protoporphyrin monomethylester (MgProtoME) cyclase reaction. The impairment of tetrapyrrole biosynthesis was examined by separate inactivation of both cyclase encoding genes followed by analysis of chlorophyll contents, MgProtoME levels and several enzyme activities of tetrapyrrole biosynthesis. We additionally addressed the question, whether the two isoforms can complement cyclase deficiency under normal aerobic and micro-oxic growth conditions in light. A cycII knock-out mutant grew without any adverse symptoms at normal air conditions, but showed MgProtoME accumulation at growth under low oxygen conditions. A complete deletion of cycI failed in spite of mixotrophic growth and low light at both ambient and low oxygen, but resulted in accumulation of 150 and 28 times more MgProtoME, respectively, and circa 60% of the wild-type chlorophyll content. The CycI deficiency induced a feedback-controlled limitation of the metabolic flow in the tetrapyrrole biosynthetic pathway by reduced ALA synthesis and Fe chelatase activity. Ectopic expression of the CycI protein restored the wild-type phenotype in cycI− mutant cells under ambient air as well as micro-oxic growth conditions. Overexpressed CycII protein could not compensate for cycI− mutation under micro-oxic and aerobic growth conditions, but complemented the cycII knock-out mutant as indicated by wild-type MgProtoME and chlorophyll levels. Our findings indicate the essential contribution of CycI to the cyclase reaction at ambient and low oxygen conditions, while low oxygen conditions additionally require CycII for the cyclase activity.

NADPH-dependent thioredoxin reductase and 2-Cys peroxiredoxins are needed for the protection of Mg–protoporphyrin monomethyl ester cyclase

The chloroplast-localized NADPH-dependent thioredoxin reductase (NTRC) has been found to be able to reduce hydrogen peroxide scavenging 2-Cys peroxiredoxins. We show that the Arabidopsis ntrc mutant is perturbed in chlorophyll biosynthesis and accumulate intermediates preceding protochlorophyllide formation. A specific involvement of NTRC during biosynthesis of protochlorophyllide is indicated from in vitro aerobic cyclase assays in which the conversion of Mg–protoporhyrin monomethyl ester into protochlorophyllide is stimulated by addition of the NTRC/2-Cys peroxiredoxin system. These findings support the hypothesis that this NADPH-dependent hydrogen peroxide scavenging system is particularly important during periods with limited reducing power from photosynthesis, e.g. under chloroplast biogenesis.

Global Regulation of Photosynthesis and Respiration by FnrL: THE FIRST TWO TARGETS IN THE TETRAPYRROLE PATHWAY

Fnr is a regulator that controls the expression of a variety of genes in response to oxygen limitation in bacteria. To assess the role of Fnr in photosynthesis in Rubrivivax gelatinosus, a strain carrying a null mutation in fnrL was constructed. It was unable to grow anaerobically in the light, but, intriguingly, it was able to produce photosynthetic complexes under high oxygenation conditions. The mutant lacked all c-type cytochromes normally detectable in microaerobically-grown wild type cells and accumulated coproporphyrin III. These data suggested that the pleiotropic phenotype observed in FNR is primarily due to the control at the level of the HemN oxygen-independent coproporphyrinogen III dehydrogenase. hemN expression in trans partially suppressed the FNR phenotype, as it rescued heme and cytochrome syntheses. Nevertheless, these cells were photosynthetically deficient, and pigment analyses showed that they were blocked at the level of Mg(2+)-protoporphyrin monomethyl ester. Expression of both hemN and bchE in the FNR mutant restored synthesis of Mg(2+)-protochlorophyllide. We, therefore, conclude that FnrL controls respiration by regulating hemN expression and controls photosynthesis by regulating both hemN and bchE expression. A comprehensive picture of the control points of microaerobic respiration and photosynthesis by FnrL is provided, and the prominent role of this factor in activating alternative gene programs after reduction of oxygen tension in facultative aerobes is discussed.

Cloning and expression of the tobacco CHLM sequence encoding Mg protoporphyrin IX methyltransferase and its interaction with Mg chelatase

S-adenosyl-L-methionine:Mg-protoporphyrin IX methyltransferase (MgPMT) is an enzyme in the Mg branch of the tetrapyrrole biosynthetic pathway. The nucleotide sequence of tobacco (Nicotiana tabacum) CHLM was identified and the cDNA sequence was used to express the precursor, the mature and a truncated recombinant MgPMT for enzymatic activity tests and for the formation of polyclonal antibodies. Comparison of the mature and the truncated MgPMT revealed three critical amino acids at the N-terminus of MgPMT for the maintenance of enzyme activity. To assess the contribution of CHLM expression to the control of the metabolic flow in the tetrapyrrole pathway, CHLM transcripts and protein levels, the enzyme activity and the steady-state levels of Mg protoporphyrin and Mg protoporphyrin monomethylester were analysed during greening of seedlings and plant development as well as under day/night and continuous growth conditions. These expression studies revealed posttranslational activation of MgPMT during greening and light/dark-cycles. Using the yeast two-hybrid system physical interaction was demonstrated between MgPMT and the CHLH subunit of Mg chelatase. Activity of recombinant MgPMT expressed in yeast cells was stimulated in the presence of the recombinant CHLH subunit. Implications for posttranslational regulation of MgPMT are discussed for the enzymatic steps at the beginning of the Mg branch.

The greening process in cress seedlings. V. Possible interference of chlorophyll precursors, accumulated after thujaplicin treatment, with light-regulated expression of Lhc genes

Dark-grown cress seedlings were treated with β-thujaplicin alone or in combination with 5-aminolevulinate (ALA). This treatment induced a nearly complete inhibition of protochlorophyllide (Pchlide) synthesis and a significant accumulation of magnesium protoporphyrin monomethylester (MgPPMe). Other porphyrins accumulated only in 48-h-old seedlings but not in 72-h-old seedlings. Levels of several mRNAs were differentially affected by pretreatment. The level of psbA and POR transcripts was either not affected in the dark (48-h-old seedlings) or only slightly decreased (72-h-old seedlings) by thujaplicin treatment. The moderate increase of the psbA transcript level by farred light was partly inhibited by thujaplicin in 48-h and 72-h-old seedlings. The level of the Lhc mRNA was increased by thujaplicin treatment in the dark in 48-h-old seedlings, comparable with the level achieved by light induction in the water controls. In contrast, the Lhc mRNA level in 72-h-old seedlings was slightly decreased by thujaplicin and the subsequent light induction was inhibited by about 50%. This inhibition of light induction was found for all investigated single Lhc genes. Differential interference by Chl precursors other than Pchlide with Lhc gene expression is discussed.

The bacteriochlorophyll biosynthesis gene, bchM, of Rhodobacter sphaeroides encodes S-adenosyl- L-methionine: Mg protoporphyrin IX methyltransferase

The bchM gene of Rhodobacter sphaeroides has been sequenced and then overexpressed in E. coli producing a protein of M r approximately 27,500. Cell-free extracts of the transformed E. coli strain are able to methylate added Mg protoporphyrin, resulting in the formation of Mg protoporphyrin monomethyl ester. The identity of this product was verified by HPLC. The bchM gene product is therefore assigned to the methyltransferase step in bacteriochlorophyll biosynthesis.

Uptake of protoporphyrin and continuous spectrophotometric assay for magnesium chelatase in Rhodobacter spheroides.

Uptake of protoporphyrin was shown by Rhodobacter spheroides under anaerobic conditions in the dark. The process was not energy-dependent but required EGTA and was markedly stimulated by Methyl Viologen. Kinetic studies were consistent with a saturable process with Kd = 5-10 microM and Bmax. = 2.2 nmol of protoporphyrin bound/mg dry weight of cells. Bound protoporphyrin could be converted into magnesium protoporphyrin monomethyl ester under anaerobic conditions in the light or at low pO2 (6.3%) in the dark. This formed the basis of a sensitive continuous spectrophotometric assay for magnesium chelatase, which avoids the need to extract the product into organic solvent, and may facilitate the development of a cell-free system for magnesium chelatase in photosynthetic bacteria. It is proposed also that the uptake mechanism shown for exogenous protoporphyrin may indicate the existence of a ligand or carrier system for endogenously produced protoporphyrin essential for magnesium chelatase activity.

Gene expression and control of enzymes for synthesis of magnesium protoporphyrin monomethyl ester in Rhodobacter sphaeroides.

Gene expression and control of enzymes for synthesis of magnesium protoporphyrin monomethyl ester in Rhodobacter sphaeroides.

The Greening Process in Cress Seedlings. III. Age-Dependent Changes in the Capacity of the Tetrapyrrole Pathway

Accumulation of protochlorophyllide (Pchlide) was determined after incubation of etiolated seedlings of garden cress (Lcpidium sativum L.) with exogenous 5-aminolevulinate for 24 h. Young seedlings (start of incubation 0-12 h after imbibition) accumulated only moderate amounts (<0.2 nmol per seedling) of Pchlide. Maximum capacity of Pchlide accumulation (ca. 14 nmol per seedling) was reached at 30-36 h after imbibition. At 60-72 h after imbibition, the capacity of Pchlide formation from exogenous ALA was only 0.2-0.3 nmol per seedling. Uptake of ALA via roots was high in young and old seedlings. The carotenoid content and pattern did not change by ALA incubation. Precursors of Pchlide were also accumulated by ALA treatment. Predominant products were protoporphyrin and magnesium protoporphyrin (MgP) in 30 h old seedlings but magnesium protoporphyrin monomethylester (MgPMe) in 72 h old seedlings. The previously described interaction of Pchlide precursors with cab gene expression cannot be correlated with a single porphyrin. A possible correlation with the ratio MgPMe: MgP is discussed.

Control of cab gene expression in synchronized Chlamydomonas reinhardtii cells

In light—dark synchronized Chlamydomonas reinhardtii cultures transcripts of at least two members of the cab gene family coding for chlorophyll a/b binding proteins are highly abundant in the light, but almost undetectable in the dark. “Run-on” transcription assays in isolated nuclei were used to show that the rapid increase in cab mRNA levels during the light phase is primarily due to regulation at the transcriptional level. Functionally unrelated inhibitors such as dipyridyl and cycloheximide as well as anaerobic conditions block chlorophyll synthesis, presumably by interfering with the conversion of magnesium protoporphyrin monomethyl ester to protochlorophyllide. Under these conditions, cab mRNA does not accumulate and nuclei isolated from inhibitor-treated cells do not support cab gene transcription. Inhibitors such as dioxoheptanoic acid and diphenyl ether herbicides block earlier steps within the chlorophyll synthesis pathway without substantial effects on cab mRNA accumulation and transcription. A possible control of transcription by intermediates of the chlorophyll biosynthesis pathway is discussed.

Regulation of tetrapyrrole synthesis by light in chemostat cultures of Rhodobacter sphaeroides.

Control of bacteriochlorophyll (Bchl), magnesium protoporphyrin monomethyl ester (MgPME), cytochromes, and coproporphyrin by light was studied with chemostat cultures of Rhodobacter sphaeroides growing at a constant dilution rate. By increasing the growth-limiting light energy flux from 10 to 55 W/m2, specific Bchl contents decreased from 19.3 to 7.9 nmol/mg of protein. This was strictly proportional to a decrease in the ratio of B800-850 to B875 light-harvesting complexes. MgPME levels increased from 1.5 to 5.3 nmol/mg of protein, while cytochrome as well as coproporphyrin levels stayed constant at 0.46 and 1.95 nmol/mg of protein, respectively. Since in chemostat cultures steady-state levels of a product represent the rate of synthesis, these results infer only slight control of the rate-limiting step of total tetrapyrrol formation by light. In substrate-limited cultures MgPME was accumulated when growth and Bchl formation approached substrate saturation. This suggests that light controls a second step, i.e., MgPME conversion, whenever too much precursor is available, owing to the low sensitivity of the initial step of control. MgPME was preferentially localized in a subcellular fraction with high contents of B875 complexes. A second fraction exhibiting increased contents of B800-850 complexes lacked significant levels of MgPME. These results are discussed in terms of localization of Bchl synthesis in the membrane system of R. sphaeroides.

The use of continuous assays to characterize the oxidative cyclase that synthesizes the chlorophyll isocyclic ring

A continuous spectroscopic assay has been developed for magnesium protoporphyrin monomethyl ester oxidative cyclase, which records either the dark formation of both free and protein-bound magnesium phaeoporphyrin or, following flash illumination, its corresponding chlorin. The properties of the enzyme were studied in wheat etioplasts. When plastids were pre-illuminated in the presence of NADPH all endogenous protochlorophyllide was converted into chlorophyllide and the product of dark incubation with magnesium protoporphyrin monomethyl ester was protein-bound magnesium 2-vinyl phaeoporphyrin a5 monomethyl ester with either a vinyl or an ethyl group at position 4 of the macrocycle alone. Rates of chlorin production from magnesium protoporphyrin monomethyl ester (up to 1240 pmol/h per mg of protein) were adequate to support known rates of plant chlorophyll synthesis. The enzyme required NADPH and O2 and had an approximate Km of 0.5 microM for magnesium protoporphyrin IX monomethyl ester. Lipid-soluble metal-complexing agents inhibited enzyme activity: hydrophilic agents were ineffective. The strong inhibition of mycobactin suggested the involvement of iron ions. Zinc protoporphyrin monomethyl ester, but not copper or nickel or metal-free protoporphyrin monomethyl esters, was a substrate; magnesium protoporphyrin dimethyl ester was inhibitory. The activity of the enzyme was unchanged by prior greening of the plants. The activity in isolated etioplasts was very dependent upon intactness of the plastid structure.

Metabolism of Magnesium Protoporphyrin Monomethyl Ester in Chlamydomonas reinhardtii

The y-1 mutant of Chlamydomonas reinhardtii is defective in the conversion of protochlorophyllide (Pchlide) to chlorophyllide in the dark. Aerobic delta-aminolevulinic acid (ALA) feeding of y-1 cells causes protoporphyrin monomethyl ester (PME) to accumulate in addition to increased levels of Pchlide. y-1 cell homogenates are not capable of methylating protoporphyrin (PROTO) to form PME but can methylate magnesium protoporphyrin (MgP) to form magnesium protoporphyrin monomethyl ester (MgPME). Anaerobic ALA feeding of y-1 causes concomitant accumulation of PME and MgPME. y-1 cells treated with alpha,alpha'-dipyridyl (DP) accumulate MgPME but not PROTO or PME. A mutant strain (bme) of Chlamydomonas has been isolated which has very little chlorophyll and accumulates PME. bme Cell homogenates can methylate MgP but not PROTO. We propose that: (a) in Chlamydomonas, PME is the initial breakdown product of MgPME; (b) both the breakdown of MgPME to PME and the conversion of MgPME to Pchlide require O(2); (c) the breakdown of MgPME to PME appears to require Fe; and (d) the PME accumulated in the bme mutant is the result of an increased breakdown of MgPME.

Studies of chloroplast development in four maize mutants defective in chlorophyll biosynthesis

Four mutants of maize (Zea mays L.) defective in chlorophyll biosynthesis have been analyzed with regard to the sites of their lesions and their effects on chloroplast development. Two yellow mutants, which accumulate no detectable porphyrin precursors when grown in darkness, are defective in the conversion of protoporphyrin IX to magnesium protoporphyrin. Etioplasts of these mutants may develop elaborate lamellar membrane systems, but prolamellar bodies are never observed. Two mutants, which are necrotic when grown under illumination, develop normal (non-necrotic) leaf tissue in the dark and accumulate a small amount of magnesium protoporphyrin monomethyl ester, corresponding approximately to the amount of protochlorophyllide accumulated by normal plants. The etioplasts of these mutants contain noncrystalline bodies. The implications of these observations with respect to chloroplast development are discussed.