Cerium Relieves the Inhibition of Chlorophyll Biosynthesis of Maize Caused by Magnesium Deficiency

Lanthanoids (Ln) were demonstrated to improve chlorophyll formation and the growth of plants. But the mechanism of the fact that Ln promotes chlorophyll biosynthesis of plants is poorly understood. The main aim of the study was to determine Ln effects in chlorophyll formation of maize under magnesium (Mg) deficiency. Maize plants were cultivated in Hoagland’s solution. They were subjected to Mg deficiency and to cerium administered in Mg-deficient Hoagland’s media, and then the contents of various chlorophyll precursors and gen expressions of the key enzymes of chlorophyll biosynthesis were examined. The decrease of chlorophyll contents in maize leaves caused by Mg deficiency suggested an inhibition of chlorophyll synthesis that was inhibited by a reduction of the precursors as measured by analyzing the contents of δ-aminolevulinic acid, porphobilinogen, uroporphyrinogen III, Mg–protoporphyrin IX, and protochlorophyll, as well as the expression levels of magnesium chelatase, magnesium-protoporphyrin IX methyltransferase, and chlorophyll synthase; Mg deficiency significantly inhibited the transformation from coproporphyrinogen III or protoporphyrin IX to chlorophyll. However, cerium addition significantly relieved the inhibition of chlorophyll biosynthesis in maize caused by Mg deficiency and increased chlorophyll content and promoted a series of transformations from δ-aminolevulinic acid to chlorophyll and maize growth under Mg deficiency. It implied that cerium might partly substitute for the role of Mg.     

Suppression of both ELIP1 and ELIP2 in Arabidopsis does not affect tolerance to photoinhibition and photooxidative stress

ELIPs (early light-induced proteins) are thylakoid proteins transiently induced during greening of etiolated seedlings and during exposure to high light stress conditions. This expression pattern suggests that these proteins may be involved in the protection of the photosynthetic apparatus against photooxidative damage. To test this hypothesis, we have generated Arabidopsis (Arabidopsis thaliana) mutant plants null for both elip genes (Elip1 and Elip2) and have analyzed their sensitivity to light during greening of seedlings and to high light and cold in mature plants. In particular, we have evaluated the extent of damage to photosystem II, the level of lipid peroxidation, the presence of uncoupled chlorophyll molecules, and the nonphotochemical quenching of excitation energy. The absence of ELIPs during greening at moderate light intensities slightly reduced the rate of chlorophyll accumulation but did not modify the extent of photoinhibition. In mature plants, the absence of ELIP1 and ELIP2 did not modify the sensitivity to photoinhibition and photooxidation or the ability to recover from light stress. This raises questions about the photoprotective function of these proteins. Moreover, no compensatory accumulation of other ELIP-like proteins (SEPs, OHPs) was found in the elip1/elip2 double mutant during high light stress. elip1/elip2 mutant plants show only a slight reduction in the chlorophyll content in mature leaves and greening seedlings and a lower zeaxanthin accumulation in high light conditions, suggesting that ELIPs could somehow affect the stability or synthesis of these pigments. On the basis of these results, we make a number of suggestions concerning the biological function of ELIPs.     

Modulation of chlorophyll biosynthesis by water stress in rice seedlings during chloroplast biogenesis*

To understand the impact of water stress on the greening process, water stress was applied to 6-day-old etiolated seedlings of a drought-sensitive cultivar of rice (Oryza sativa), Pusa Basmati-1 by immersing their roots in 40 mm polyethylene glycol (PEG) 6000 (-0.69 MPa) or 50 mm PEG 6000 (-1.03 MPa) dissolved in half-strength Murashige and Skoog (MS)-nutrient-solution, 16 h prior to transfer to cool-white-fluorescent + incandescent light. Chlorophyll (Chl) accumulation substantially declined in developing water-stressed seedlings. Reduced Chl synthesis was due to decreased accumulation of chlorophyll biosynthetic intermediates, that is, glutamate-1-semialdehyde (GSA), 5-aminolevulinic acid, Mg-protoporphyrin IX monomethylester and protochlorophyllide. Although 5-aminolevulinic acid synthesis decreased, the gene expression and protein abundance of the enzyme responsible for its synthesis, GSA aminotransferase, increased, suggesting its crucial role in the greening process in stressful environment. The biochemical activities of Chl biosynthetic enzymes, that is, 5-aminolevulinic acid dehydratase, porphobilinogen deaminase, coproporphyrinogen III oxidase, porphyrinogen IX oxidase, Mg-chelatase and protochlorophyllide oxidoreductase, were down-regulated due to their reduced protein abundance/gene expression in water-stressed seedlings. Down-regulation of protochlorophyllide oxidoreductase resulted in impaired Shibata shift. Our results demonstrate that reduced synthesis of early intermediates, that is, GSA and 5-aminolevulinic acid, could modulate the gene expression of later enzymes of Chl biosynthesis pathway.     

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 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.     

Impaired expression of the plastidic ferrochelatase by antisense RNA synthesis leads to a necrotic phenotype of transformed tobacco plants

Protoporphyrin IX is the last common intermediate of tetrapyrrole biosynthesis. The chelation of a Mg2+ ion by magnesium chelatase and of a ferrous ion by ferrochelatase directs protoporphyrin IX towards the formation of chlorophyll and heme, respectively. A full length cDNA clone encoding a ferrochelatase was identified from a Nicotiana tabacum cDNA library. The encoded protein consists of 497 amino acid residues with a molecular weight of 55.4 kDa. In vitro import of the protein into chloroplasts and its location in stroma and thylakoids confirm its close relationship to the previously described Arabidopsis thaliana plastid-located ferrochelatase (FeChII). A 1700-bp tobacco FeCh cDNA sequence was expressed in Nicotiana tabacum cv. Samsun NN under the control of the CaMV 35S promoter in antisense orientation allowing investigation into the consequences of selective reduction of the plastidic ferrochelatase activity for protoporphyrin IX channeling in chloroplasts and for interactions between plastidic and mitochondrial heme synthesis. Leaves of several transformants showed a reduced chlorophyll content and, during development, a light intensity-dependent formation of necrotic leaf lesions. In comparison with wild-type plants the total ferrochelatase activity was decreased in transgenic lines leading to an accumulation of photosensitizing protoporphyrin IX. Ferrochelatase activity was reduced only in plastids but not in mitochondria of transgenic plants. By means of the specifically diminished ferrochelatase activity consequences of the selective inhibition of protoheme formation for the intracellular supply of heme can be investigated in the future.     

The effect of simulated microgravity on formation of the pigment apparatus in etiolated barley seedlings

The effect of horizontal clinorotation on the dynamics of the accumulation of the main photosynthetic pigments in the greening of 6-day-old etiolated barley seedlings has been studied. The content of protochlorophillide, the direct precursor of chlorophyll a, in clinorotated seedlings in the dark was 9–20% lower than in the control group. After exposure of barley seedlings to light for 12 h under clinorotation, chlorophyll accumulation lagged behind the control by 45% and reached the control value after 48–72 h. The total content of carotenoids increased many fold during greening; at the first stage the carotenoid level in clinorotated seedlings was less than in the control. The synthesis rates of δ-aminolevulinic acid and δ-aminolevulinate dehydratase activity in clinorotated seedlings were slower than in the control after 24 h of greening and after 72 h of greening reaching the control values. The activity of Mg-protoporphyrin IX chelatase catalyzing the incorporation of Mg ions in the structure of chlorophyll a, did not change when exposed to clinorotation. The results we obtained show inhibition of the initial stages of chlorophyll biosynthesis in the conditions of simulated microgravity. The light, to a certain extent, decreases the negative effect of microgravity on the formation of the photosynthetic apparatus in plants.     

Selenium as a novel regulator of porphyrin biosynthesis in germinating seedlings of mung bean (Phaseolus vulgaris)

5-Aminolevulinic acid, porphyrin and chlorophyll contents as well the activities of 5-aminolevulinic acid dehydratase and PBG deaminase were studied in selenium treated mung bean seedlings. Selenium had no effect on 5-aminolevulinic acid synthetic ability but inhibited 5-aminolevulinic acid dehydratase and PBG deaminase activities. Further, it was observed that selenium induced accumulation of protoporphyrin-IX and Mg-protoporphyrin ester and decreased chlorophyll levels in both light and dark-grown seedlings. The results suggest the possible regulatory role of selenium on chlorophyll synthesis by interacting with sulfhydryl containing enzymes 5-aminolevulinic acid dehydratase and porphobilinogen deaminase.     

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     

Light control of porphyrin accumulation in acifluorfen-methyl-treated Lemna pausicostata

In Lemna pausicostata Hegelm. 6746, light is required for sufficient acifluorfenmethyl (AFM) stimulation of protoporphyrin IX (Proto IX) accumulation to cause significant herbicidal action. In darkness, AFM causes Proto IX levels to increase for about 2 h, after which Proto IX content is stable at levels significantly lower than those accumulated in light. In darkness, sucrose cannot increase levels of AFM-induced Proto IX. However, addition of δ-aminolevulinic acid (ALA) increases Proto IX levels in AFM-treated plants in darkness, demonstrating that the herbicide blocks the porphyrin pathway in darkness as it does in the light. Thus, Proto IX accumulation in darkness appears to be limited by ALA availability. This is supported by the finding that dioxoheptanoic acid caused more ALA to accumulate in light than in darkness. Heme is a feedback inhibitor of ALA synthesis, and heme synthesis is inhibited by AFM. However, total extractable heme levels were reduced by AFM by about the same amount in both light and darkness. Exogenously supplied hemin reduced AFM-caused Proto IX accumulation and herbicidal damage in the light and also reduced Proto IX accumulation caused by AFM or AFM plus ALA in darkness. AFM-stimulated Proto IX accumulation was inversely proportional to the log of the photon flux density between 5 and 500 μmol in m⁻² s⁻¹. Reduced effects of higher photon fluxes on AFM-stimulated Proto IX accumulation are probably due to both increased photobleaching of Proto IX and reduced porphyrin synthesis because of herbicidal damage. AFM-stimulated Proto IX accumulation in darkness could not be demonstrated to be under phytochrome control, but it appeared to be under the negative influence of protochlorophyllide levels.     

Growth Cycle-Dependent Overproduction and Accumulation of Protoporphyrin IX in Tetrahymena: Effect of Heavy Metals1

Cells of the ciliate Tetrahymena pyriformis GL overproduce and accumulate massive quantities of the heme intermediate, protoporphyrin IX. Protoporphyrin is localized intracellularly in discrete membranous compartments. The amount of porphyrin stored in the cell changes dramatically as cells progress through the growth cycle. Porphyrin overproduction is stimulated by δ-aminolevulinic acid, but only during the mid-stationary phase. Overproduction of protoporphyrin IX apparently results from an increase, late in the growth cycle, of activities subsequent to δ-aminolevulinic acid synthetase. Feedback inhibition in the pathway by accumulated protoporphyrin IX does not occur. The presence of Co²⁺ completely inhibits accumulation of protoporphyrin IX in a manner reversed by δ-aminolevulinic acid. Sn⁴⁺ stimulates protoporphyrin IX accumulation in the culture.     

The C-terminal extension of ferrochelatase is critical for enzyme activity and for functioning of the tetrapyrrole pathway in Synechocystis strain PCC 6803

Heme and chlorophyll (Chl) share a common biosynthetic pathway up to the branch point where magnesium chelatase and ferrochelatase (FeCH) insert either magnesium for Chl biosynthesis or ferrous iron for heme biosynthesis. A distinctive feature of FeCHs in cyanobacteria is their C-terminal extension, which forms a putative transmembrane segment containing a Chl-binding motif. We analyzed the ΔH324 strain of Synechocystis sp. strain PCC 6803, which contains a truncated FeCH enzyme lacking this C-terminal domain. Truncated FeCH was localized to the membrane fraction, suggesting that the C-terminal domain is not necessary for membrane association of the enzyme. Measurements of enzyme activity and complementation experiments revealed that the ΔH324 mutation dramatically reduced activity of the FeCH, which resulted in highly upregulated 5-aminolevulinic acid synthesis in the ΔH324 mutant, implying a direct role for heme in the regulation of flux through the pathway. Moreover, the ΔH324 mutant accumulated a large amount of protoporphyrin IX, and levels of Chl precursors were also significantly increased, suggesting that some, but not all, of the “extra” flux can be diverted down the Chl branch. Analysis of the recombinant full-length and truncated FeCHs demonstrated that the C-terminal extension is critical for activity of the FeCH and that it is strictly required for oligomerization of this enzyme. The observed changes in tetrapyrrole trafficking and the role of the C terminus in the functioning of FeCH are discussed.     

Inactivation of Mg chelatase during transition from anaerobic to aerobic growth in Rhodobacter capsulatus

The facultative photosynthetic bacterium Rhodobacter capsulatus can adapt from an anaerobic photosynthetic mode of growth to aerobic heterotrophic metabolism. As this adaptation occurs, the cells must rapidly halt bacteriochlorophyll synthesis to prevent phototoxic tetrapyrroles from accumulating, while still allowing heme synthesis to continue. A likely control point is Mg chelatase, the enzyme that diverts protoporphyrin IX from heme biosynthesis toward the bacteriochlorophyll biosynthetic pathway by inserting Mg(2+) to form Mg-protoporphyrin IX. Mg chelatase is composed of three subunits that are encoded by the bchI, bchD, and bchH genes in R. capsulatus. We report that BchH is the rate-limiting component of Mg chelatase activity in cell extracts. BchH binds protoporphyrin IX, and BchH that has been expressed and purified from Escherichia coli is red in color due to the bound protoporphyrin IX. Recombinant BchH is rapidly inactivated by light in the presence of O(2), and the inactivation results in the formation of a covalent adduct between the protein and the bound protoporphyrin IX. When photosynthetically growing R. capsulatus cells are transferred to aerobic conditions, Mg chelatase is rapidly inactivated, and BchH is the component that is most rapidly inactivated in vivo when cells are exposed to aerobic conditions. The light- and O(2)-stimulated inactivation of BchH could account for the rapid inactivation of Mg chelatase in vivo and provide a mechanism for inhibiting the synthesis of bacteriochlorophyll during adaptation of photosynthetically grown cells to aerobic conditions while still allowing heme synthesis to occur for aerobic respiration.     

Physiological basis for differential sensitivities of plant species to protoporphyrinogen oxidase-inhibiting herbicides

With a leaf disc assay, 11 species were tested for effects of the herbicide acifluorfen on porphyrin accumulation in darkness and subsequent electrolyte leakage and photobleaching of chlorophyll after exposure to light. Protoporphyrin IX (Proto IX) was the only porphyrin that was substantially increased by the herbicide in any of the species. However, there was a wide range in the amount of Proto IX accumulation caused by 0.1 millimolar acifluorfen between species. Within species, there was a reduced effect of the herbicide in older tissues. Therefore, direct quantitative comparisons between species are difficult. Nevertheless, when data from different species and from tissues of different age within a species were plotted, there was a curvilinear relationship between the amount of Proto IX caused to accumulate during 20 hours of darkness and the amount of electrolyte leakage or chlorophyll photobleaching caused after 6 and 24 hours of light, respectively, following the dark period. Herbicidal damage plateaued at about 10 nanomoles of Proto IX per gram of fresh weight. Little difference was found between in vitro acifluorfen inhibition of protoporphyrinogen oxidase (Protox) of plastid preparations of mustard, cucumber, and morning glory, three species with large differences in their susceptibility at the tissue level. Mustard, a highly tolerant species, produced little Proto IX in response to the herbicide, despite having a highly susceptible Protox. Acifluorfen blocked carbon flow from δ-aminolevulinic acid to protochlorophyllide in mustard, indicating that it inhibits Protox in vivo. Increasing δ-aminolevulinic acid concentrations (33-333 micromolar) supplied to mustard with 0.1 millimolar acifluorfen increased Proto IX accumulation and herbicidal activity, demonstrating that mustard sensitivity to Proto IX was similar to other species. Differential susceptibility to acifluorfen of the species examined in this study appears to be due in large part to differences in Proto IX accumulation in response to the herbicide. In some cases, differences in Proto IX accumulation appear to be due to differences in activity of the porphyrin pathway.     

4-amino-5-hexynoic Acid-a potent inhibitor of tetrapyrrole biosynthesis in plants

4-Amino-5-hexynoic acid, a suicide inactivator of the mammalian pyridoxal phosphate-dependent 4-aminobutyric acid:2-oxoglutaric acid aminotransferase, inhibits phytochrome and chlorophyll synthesis in developing oat (Avena sativa L.), corn (Zea mays L.), pea (Pisum sativum L.), and cucumber (Cucumis sativus L.) seedlings. In Avena and Cucumis seedlings, respectively, inhibition of phytochrome and chlorophyll accumulation by 4-amino-5-hexynoic acid can be significantly reversed by application of 5-aminolevulinic acid. These results indicate that 4-amino-5-hexynoic acid inhibits the synthesis of 5-aminolevulinic acid in plants.     

Expression of ELIPs and PS II-S protein in spinach during acclimative reduction of the Photosystem II antenna in response to increased light intensities

The PS II-S protein and the so-called early light-inducible proteins (ELIPs) are homologous to the chlorophyll a/b-binding (Cab) gene products functioning in light-harvesting. The functional significance of these two CAB homologues is not known although they have been considered to bind pigments and in the case of the PS II–S protein this has been experimentally supported. The role of these two proteins does not appear to be light-harvesting but instead they are suggested to play a role as quenchers of free chlorophyll molecules during biogenesis and/or degradation of pigment-binding proteins. Such a role would be essential to eliminate the toxic and damaging effects that can be induced by free chlorophyll in the light. To this end the expression and characteristics of the ELIPs and the PS II–S protein were investigated in spinach leaves acclimating from low to high light intensities. Under these conditions there is a reduction in the antenna size of Photosystem II due to proteolytic digestion of its major chlorophyll a/b-binding protein (LHC II). During this acclimative proteolysis, up to one third of LHC II can be degraded and consequently substantial amounts of chlorophyll molecules will lose their binding sites. Our results reveal that there is a close correlation between ELIP accumulation and the onset of the LHC II degradation as low light-grown spinach leaves are subjected to increased light intensities. In contrast, there was no change in the relative level of the PS II–S protein during the acclimation process. It is concluded that the role for the ELIPs may be related to binding of liberated chlorophyll molecules and quenching of the toxic effects during LHC II degradation. In addition it was shown that in spinach four different ELIP species can be expressed and that they show different accumulation patterns in response to increased light intensities.