Effects of Cerium on Key Enzymes of Carbon Assimilation of Spinach Under Magnesium Deficiency

The mechanism of the fact that cerium improves the photosynthesis of plants under magnesium deficiency is poorly understood. The main aim of the study was to determine the role of cerium in the amelioration of magnesium deficiency effects in CO₂ assimilation of spinach. Spinach plants were cultivated in Hoagland’s solution. They were subjected to magnesium deficiency and to cerium chloride administered in the magnesium-present Hoagland’s media and magnesium-deficient Hoagland’s media. The results showed that the chlorophyll synthesis and oxygen evolution was destroyed, and the activities of Rubisco carboxylasae and Rubisco activase and the expression of Rubisco large subunit (rbcL), Rubisco small subunit (rbcS), and Rubisco activase subunit (rca) were significantly inhibited, then plant growth was inhibited by magnesium deficiency. However, cerium promotes the chlorophyll synthesis, the activities of two key enzymes in CO₂ assimilation, and the expression of rbcL, rbcS, and rca, thus leading to the enhancement of spinach growth under magnesium-deficient conditions.     

Improvement of Cerium of Photosynthesis Functions of Maize Under Magnesium Deficiency

Rare earth elements can promote photosynthesis, but their mechanisms are still poorly understood under magnesium deficiency. The present study was designed to determine the role of cerium in magnesium-deficient maize plants. Maize was cultivated in Hoagland’s solution added with cerium with and without adequate quantities of magnesium. Under magnesium-deficient conditions, cerium can prevents inhibition of synthesis of photosynthetic pigment, improves light energy absorption and conversion, oxygen evolution, and the activity of photo-phosphorelation and its coupling factor Ca²⁺-ATPase. These results suggest that cerium could partly substitute magnesium, improving photosynthesis and plant growth.     

An analysis of precursors accumulated by several chlorophyll biosynthetic mutants of maize

Summary Several mutants of maize defective in chlorophyll synthesis are analysed. By feeding shoots of dark-grown seedlings -aminolevulinic acid, the regulatory step in chlorophyll biosynthesis is bypassed and chlorophyll precursors accumulate. In normal plants this results in a buildup of protoporphyrin IX and protochlorophyllide, while mutants accumulate precursors, depending on the site of the mutant-induced lesion. Mutants at three loci, l ^*-Blandy4, 113, and oy, are defective in conversion of protoporphyrin IX to Mg-protoporphyrin. Mutants at the oro and oro2 loci are defective in conversion of Mg-protoporphyrin monomethyl ester to protochlorophyllide. A dominant modifier gene, Orom, which allows oro seedlings to bypass their lesion is also described.Journal Paper No J-9076 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa Project No. 2035     

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.     

The light stress-induced protein ELIP2 is a regulator of chlorophyll synthesis in Arabidopsis thaliana

The early light-induced proteins (ELIPs) belong to the multigenic family of pigment-binding light-harvesting complexes. ELIPs accumulate transiently and are believed to play a protective role in plants exposed to high levels of light. Constitutive expression of the ELIP2 gene in Arabidopsis resulted in a marked reduction of the pigment content of the chloroplasts, both in mature leaves and during greening of etiolated seedlings. The chlorophyll loss was associated with a decrease in the number of photosystems in the thylakoid membranes, but the photosystems present were fully assembled and functional. A detailed analysis of the chlorophyll-synthesizing pathway indicated that ELIP2 accumulation downregulated the level and activity of two important regulatory steps: 5-aminolevulinate synthesis and Mg-protoporphyrin IX (Mg-Proto IX) chelatase activity. The contents of glutamyl tRNA reductase and Mg chelatase subunits CHLH and CHLI were lowered in response to ELIP2 accumulation. In contrast, ferrochelatase activity was not affected and the inhibition of Heme synthesis was null or very moderate. As a result of reduced metabolic flow from 5-aminolevulinic acid, the steady state levels of various chlorophyll precursors (from protoporphyrin IX to protochlorophyllide) were strongly reduced in the ELIP2 overexpressors. Taken together, our results indicate that the physiological function of ELIPs could be related to the regulation of chlorophyll concentration in thylakoids. This seems to occur through an inhibition of the entire chlorophyll biosynthesis pathway from the initial precursor of tetrapyrroles, 5-aminolevulinic acid. We suggest that ELIPs work as chlorophyll sensors that modulate chlorophyll synthesis to prevent accumulation of free chlorophyll, and hence prevent photooxidative stress.     

Growth of Zinc-Deficient Rats During Intra-Gastric Tube Feeding

The main aim of the study was to determine the role of cerium in the amelioration of calcium-deficiency effects in spinach plants. Spinach plants were cultivated in Hoagland’s solution. They were subjected to calcium-deficiency and to cerium chloride administered in the calcium-present Hoagland’s media and calcium-deficient Hoagland’s media. Within 3weeks, young leaves developed distinct calcium-deficient symptoms, and plant growth significantly inhibited to calcium deprivation as would be expected; cerium-treated groups grown in the same conditions did not develop calcium-deficient symptoms; fresh weight, dry weight and chlorophyll content of spinach plants were increased by 35.9, 45 and 64.05% compared to those of plants cultivated in calcium-deficient media. In addition, calcium deprivation in spinach plants caused the reduction of photosynthetic rate, oxygen evolution rate and ribulose-1,5-bisphosphate carboxylase/oxygenase activity. The reduction of activities of nitrate reductase, glutamate dehydrogenase, glutamate synthase and glutamic-pyruvic transaminase was observed under calcium-deficient media. However, cerium treatment under calcium-deficient media could significantly improve photosynthesis and nitrogen metabolism of spinach plants. This is viewed as evidence that cerium added to calcium-deficient media in the spinach plants could substitute for calcium and improve spinach growth.     

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.Journal Paper No. J-9136 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa Project No. 2035     

Regulation of protoporphyrin IX biosynthesis by intraplastidic compartmentalization and adenosine triphosphate

Subplastidic preparations from cotyledons of cucumber (Cucumis sativus L.) were tested for their ability to synthesize protoporphyrin IX from the substrate 5-aminolevulinic acid. Envelope or thylakoid membranes failed to synthesize protoporphyrin IX from the substrate 5-aminolevulinic acid. Stromal preparations synthesized a very low amount of protoporphyrin IX. In a reconstitution experiment using stroma + envelope membranes, protoporphyrin IX synthesis from 5-aminolevulinic acid was enhanced by 660% over that of stroma alone. However, when thylakoids were added to the stroma + envelope mixture, protoporphyrin IX synthesis from 5-aminolevulinic acid was completely inhibited. In the reconstituted stroma + envelope membrane mixture, the reducing agent dithiothreitol enhanced the protoporphyrin IX-synthesizing ability and completely abolished the inhibition of protoporphyrin IX synthesis by thylakoids. This suggested that the oxidizing agents usually associated with the thylakoid membranes inhibited protoporphyrin IX biosynthesis and the inhibition was alleviated by the reducing power of dithiothreitol. This study exposes the weakness of in vitro reconstitution experiments in mimicking the in vivo-conditions. Addition of ATP stimulated protoporphyrin IX synthesis by 50% in the supernatant fraction of chloroplast lysate. This ATP-induced stimulation of protoporphyrin IX synthesis was due to the enhancement of the activities of uroporphyrinogen decarboxylase and protoporphyrinogen oxidase, involved in tetrapyrrole biosynthesis. The ATP-induced stimulation of porphyrinogen oxidase activity was an energy-dependent reaction. Received: 21 March 2000 / Accepted: 9 May 2000     

Immunologic evidence for structural homology between the release factors of Escherichia coli.

A cell-free chloroplast preparation obtained from greening cucumber cotyledons was tested for its ability to synthesize protoporphyrin IX from compounds previously postulated to be precursors of δ-aminolevulinic acid in plants, namely, glutamate, glutamine, α-ketoglutarate, glycine, and succinate. Of these, only glutamate caused a marked stimulation of protoporphyrin biosynthesis. A mixture of cofactors (ATP, KH₂PO₄, glutathione, coenzyme A, and NAD⁺), which was previously shown to be necessary for the incorporation of δ-aminolevulinic acid into protochlorophyll and for the maintenance of etioplasts in vitro also proved to be necessary for the conversion of glutamate to protoporphyrin IX.     

Structural and biochemical characterization of Gun4 suggests a mechanism for its role in chlorophyll biosynthesis

Gun4 has been implicated in a developmental signaling pathway between the chloroplast and the nucleus involving magnesium protoporphyrin IX (MgP(IX)), the first dedicated intermediate in the chlorophyll biosynthetic pathway. Here we present the crystal structure of Thermosynechococcus elongatus Gun4 at 1.5 A, describe the binding affinities of Gun4 for substrate and product porphyrin molecules, and identify a likely (Mg)P(IX) binding site on the protein. Kinetic analyses show that Gun4 dramatically increases the efficiency of transformation of porphyrin substrate to metalloporphyrin product and that it also reduces the threshold Mg2+ concentration required for activity at low porphyrin concentration. Gun4 therefore controls magnesium chelatase at physiologically significant Mg2+ concentrations and likely acts as a molecular switch in vivo so that in its absence magnesium chelatase is inactive. This mechanism could allow Gun4 to mediate magnesium protoporphyrin levels both for chlorophyll biosynthesis and for signaling to the nucleus.     

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.     

Regulation of Magnesium Chelatase Activity during Excessive Accumulation of Porphyrins in Green Barley Leaves

Activity of magnesium chelatase was studied in green barley leaves treated with 5-aminolevulinic acid (ALA). After this treatment, leaves accumulated excessive amounts of porphyrinic precursors of chlorophyll : protoporphyrin IX (PP), magnesium-protoporphyrin IX (MgPP), its monomethyl ester (MgPPE), and protochlorophyllide. The enzyme activity was found to be inversely dependent on the amount of MgPP formed from exogenous ALA. A conclusion was drawn about the existence of a mechanism for the regulation of the enzyme activity in vivo via its inhibition by the reaction product.     

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.     

Regulation of light-harvesting chlorophyll-binding protein mRNA accumulation in Chlamydomonas reinhardi. Possible involvement of chlorophyll synthesis precursors

Light induction of light-harvesting chlorophyll a/b-binding protein (LHCP) mRNA accumulation was studied in light-dark synchronized cultures of Chlamydomonas reinhardi. LHCP mRNA accumulation was prevented by the chlorophyll-synthesis inhibitor alpha,alpha-dipyridyl which blocks late steps in the chlorophyll biosynthetic pathway and leads to the accumulation of the porphyrin intermediate magnesium protoporphyrin methyl ester. LHCP mRNA accumulated normally, however, when chlorophyll synthesis was blocked by inhibitors such as hemin and levulinic acid which interfere with early steps in the chlorophyll biosynthesis pathway prior to the formation of magnesium protoporphyrin methyl ester. Similar effects were observed in the light induction of LHCP mRNA levels in protoporphyrin IX-accumulating mutants, brc-1 and brs-1. These mutants have low levels of LHCP mRNA when grown under heterotrophic conditions in the dark where they accumulate protoporphyrin IX. However, LHCP mRNA is light-induced in brc-1 which synthesizes chlorophyll in the light and presumably consumes porphyrin intermediates in doing so. These results suggest that the chlorophyll-synthesis intermediates, magnesium protoporphyrin methyl ester and its immediate precursors, inhibit by a feedback mechanism the light induction of LHCP mRNA accumulation. Low magnesium protoporphyrin methyl ester levels permit the light-induced accumulation of LHCP mRNA, whereas high magnesium protoporphyrin methyl ester levels destabilize LHCP mRNA regardless of the illumination conditions. Preliminary experiments show that LHCP mRNA accumulation in C. reinhardi is stimulated by blue light, and not by red light which stimulates LHCP mRNA accumulation in higher plants.     

The Maize Oil Yellow1 (Oy1) Gene Encodes the I Subunit of Magnesium Chelatase

Semi-dominant Oil yellow1 (Oy1) mutants of maize (Zea mays) are deficient in the conversion of protoporphyrin IX to magnesium protoporphyrin IX, the first committed step of chlorophyll biosynthesis. Using a candidate gene approach, a cDNA clone was isolated that was predicted to encode the I subunit of magnesium chelatase (ZmCHLI) and mapped to the same genetic interval as Oy1. Allelic variation was identified at ZmCHLI between wild-type plants and plants carrying semi-dominant alleles of Oy1. These differences revealed putative amino acid substitutions that could account for the alterations in protein function. Candidate lesions were tested by introduction of homologous changes into the Synechocystis magnesium chelatase I gene (SschlI) and characterization of the activity of mutant protein variants in an in vitro enzyme activity assay. The results of these analyses suggest that SsChlI protein variants containing the substitutions identified in the dominant Oy1 maize alleles lack activity necessary for magnesium chelation and confer a semi-dominant phenotype via competitive inhibition of wild-type SsChlI.     

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.     

The gun4 gene is essential for cyanobacterial porphyrin metabolism

Ycf53 is a hypothetical chloroplast open reading frame with similarity to the Arabidopsis nuclear gene GUN4. In plants, GUN4 is involved in tetrapyrrole biosynthesis. We demonstrate that one of the two Synechocystis sp. PCC 6803 ycf53 genes with similarity to GUN4 functions in chlorophyll (Chl) biosynthesis as well: cyanobacterial gun4 mutant cells exhibit lower Chl contents, accumulate protoporphyrin IX and show less activity not only of Mg chelatase but also of Fe chelatase. The possible role of Gun4 for the Mg as well as Fe porphyrin biosynthesis branches in Synechocystis sp. PCC 6803 is discussed.