Rice Chlorina-1 and Chlorina-9 encode ChlD and ChlI subunits of Mg-chelatase, a key enzyme for chlorophyll synthesis and chloroplast development

Photosynthetic organisms exhibit a green color due to the accumulation of chlorophyll pigments in chloroplasts. Mg-protoporphyrin IX chelatase (Mg-chelatase) comprises three subunits (ChlH, ChlD and ChlI) and catalyzes the insertion of Mg²⁺ into protoporphyrin IX, the last common intermediate precursor in both chlorophyll and heme biosyntheses, to produce Mg-protoporphyrin IX (MgProto). Chlorophyll deficiency in higher plants results in chlorina (yellowish-green) phenotype. To date, 10 chlorina (chl) mutants have been isolated in rice, but the corresponding genes have not yet been identified. Rice Chl1 and Chl9 genes were mapped to chromosome 3 and isolated by map-based cloning. A missense mutation occurred in a highly conserved amino acid of ChlD in the chl1 mutant and ChlI in the chl9 mutant. Ultrastructural analyses have revealed that the grana are poorly stacked, resulting in the underdevelopment of chloroplasts. In the seedlings fed with aminolevulinate-dipyridyl in darkness, MgProto levels in the chl1 and chl9 mutants decreased up to 25% and 31% of that in wild-type, respectively, indicating that the Mg-chelatase activity is significantly reduced, causing the eventual decrease in chlorophyll synthesis. Furthermore, Northern blot analysis indicated that the nuclear genes encoding the three subunits of Mg-chelatase and LhcpII in chl1 mutant are expressed about 2-fold higher than those in WT, but are not altered in the chl9 mutant. This result indicates that the ChlD subunit participates in negative feedback regulation of plastid-to-nucleus in the expression of nuclear genes encoding chloroplast proteins, but not the ChlI subunit.Haitao Zhang and Jinjie Li contributed equally to this work     

Heme, a plastid-derived regulator of nuclear gene expression in Chlamydomonas

To gain insight into the chloroplast-to-nucleus signaling role of tetrapyrroles, Chlamydomonas reinhardtii mutants in the Mg-chelatase that catalyzes the insertion of magnesium into protoporphyrin IX were isolated and characterized. The four mutants lack chlorophyll and show reduced levels of Mg-tetrapyrroles but increased levels of soluble heme. In the mutants, light induction of HSP70A was preserved, although Mg-protoporphyrin IX has been implicated in this induction. In wild-type cells, a shift from dark to light resulted in a transient reduction in heme levels, while the levels of Mg-protoporphyrin IX, its methyl ester, and protoporphyrin IX increased. Hemin feeding to cultures in the dark activated HSP70A. This induction was mediated by the same plastid response element (PRE) in the HSP70A promoter that has been shown to mediate induction by Mg-protoporphyrin IX and light. Other nuclear genes that harbor a PRE in their promoters also were inducible by hemin feeding. Extended incubation with hemin abrogated the competence to induce HSP70A by light or Mg-protoporphyrin IX, indicating that these signals converge on the same pathway. We propose that Mg-protoporphyrin IX and heme may serve as plastid signals that regulate the expression of nuclear genes.     

Chloroplast Biogenesis: XX. Accumulation of Porphyrin and Phorbin Pigments in Cucumber Cotyledons during Photoperiodic Greening

A study of greening in cucumber (Cucumis sativus L.) cotyledons grown under a light (14-hour) dark (10-hour) photoperiodic regime was undertaken. The pools of protoporphyrin IX, Mg-protoporphyrin IX monoester, protochlorophyllide, and protochlorophyllide ester were determined spectrofluorometrically. Chlorophyll a and b were monitored spectrophotometrically. Pigments were extracted during the 3rd hour of each light period and at the end of each subsequent dark period during the first seven growth cycles. Protoporphyrin IX did not accumulate during greening. Mg-protoporphyrin IX monoester and longer wavelength metalloporphyrins accumulated during the light cycles and disappeared in the dark. Their disappearance was accompanied by the accumulation of protochlorophyll. Higher levels of protochlorophyll were observed in the dark than in the light, and the greatest accumulation occurred during the third and fourth dark cycles. Protochlorophyllide was present in 3- to 10-fold excess over protochlorophyllide ester; it was detectable during the period of net chlorophyll accumulation as well as afterward. In contrast, protochlorophyllide ester was observable only during the first four photoperiodic cycles, suggesting that it was a metabolic intermediate only during the early stages of chlorophyll accumulation. Between the third and fourth growth cycles, a rapid increase in area and fresh weight per cotyledon began. This was accompanied by a 250-fold increase in the level of chlorophyll a + b during the three subsequent growth cycles. No lag period in the accumulation of chlorophyll b was observed, and at all stages of greening, the chlorophyll a/b ratio was approximately 3.     

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     

Chloroplast signalling in the light induction of nuclear HSP70 genes requires the accumulation of chlorophyll precursors and their accessibility to cytoplasm/nucleus

Chlorophyll precursors Mg-protoporphyrin IX and its monomethylester are candidates for plastid-derived molecules involved in light signalling from the chloroplast to the nucleus. The pool sizes of these two Mg2+-containing porphyrins and of protoporphyrin IX transiently increased upon a shift of Chlamydomonas cultures from dark to light. This increase coincided with the accumulation of mRNAs encoded by the nuclear genes HSP70A and HSP70B. Analysis of a mutant (brs-1), previously shown to be defective in the light induction of these genes, revealed high levels of protoporphyrin IX but no light-induced increase in the levels of Mg2+-containing porphyrins. Inhibitors of cytoplasmic protein synthesis prevented both the light-induced rise in pool levels and induction of the HSP70 genes. Similarly, pre-gametes, intermediates of sexual differentiation, lacked both responses to light. The block in light induction of the HSP70 genes in inhibitor-treated cells and in pre-gametes could be circumvented by the exogenous addition of Mg-protoporphyrin IX in the dark. This suggests an essential role for light-induced Mg-protoporphyrin IX accumulation in this chloroplast-to-nucleus signalling pathway. However, accumulation of this porphyrin in the dark - presumably in the chloroplast - did not result in induction. A second crucial role for light in this signalling pathway is postulated which makes this plastidic compound accessible to the cytoplasm/nucleus where the downstream signalling pathway may be activated.     

Formation of the isocyclic ring of chlorophyll by isolated Chlamydomonas reinhardtii chloroplasts

Chlamydomonas reinhardtii chloroplasts catalyzed two sequential steps of Chl biosynthesis, S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase and Mg-protoporphyrin IX monomethyl ester oxidative cyclase. A double mutant strain of C. reinhardtii was constructed which has a cell wall deficiency and is unable to form chlorophyll in the dark. Dark-grown cells were disrupted with a BioNeb nebulizer under conditions which lysed the plasma membrane but not the chloroplast envelope. Chloroplasts were purified by Percoll density gradient centrifugation. The purified chloroplasts were used to define components required for the biosynthesis of Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) from Mg-protoporphyrin IX. Product formation requires the addition of Mg-protoporphyrin IX, the substrate for S-adenosyl-l-methionine:Mg-protoporphyrin IX methyltransferase which produces Mg-protoporphyrin IX monomethyl ester. The Mg-protoporphyrin IX monomethyl ester that is generated in situ is the substrate for Mg-protoporphyrin IX monomethyl ester oxidative cyclase. The reaction product was identified as Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) by excitation and emission spectrofluorometry and HPLC on ion-paired reverse-phase and polyethylene columns. Mg-2,4-divinylpheoporphyrin a ₅ formation by the coupled enzyme system required O₂ and was stimulated by the addition of NADP⁺, an NADPH regenerating system, and S-adenosyl-l-methionine. Product was formed at a relatively steady rate for at least 60 min.Abbreviations MgDVP Mg-2,4-divinylpheoporphyrin a ₅ (divinyl protochlorophyllide) - SAM S-adenosyl-l-methionine     

Cloning and characterization of the chlorophyll biosynthesis gene chlM from Synechocystis PCC 6803 by complementation of a bacteriochlorophyll biosynthesis mutant of Rhodobacter capsulatus

A bacteriochlorophyll a biosynthesis mutant of the purple photosynthetic bacterium Rhodobacter capsulatus was functionally complemented with a cosmid genomic library from Synechocystis sp. PCC 6803. The complemented R. capsulatus strain contains a defined mutation in the bchM gene that codes for Mg-protoporphyrin IX methyltransferase, the enzyme which converts Mg-protoporphyrin IX to Mg-protoporphyrin IX methylester using S-adenosyl-l-methionine as a cofactor. Since chlorophyll biosynthesis also requires the same methylation reaction, the Synechocystis genome should similarly code for a Mg-protoporphyrin IX methyltransferase. Sequence analysis of the complementing Synechocystis cosmid indicates that it contains an open reading frame exhibiting 29% sequence identity to BchM. In addition, expression of the Synechocystis gene in the R. capsulatus bchM mutant via the strong R. capsulatus puc promoter was shown to support nearly wild-type levels of bacteriochlorophyll a synthesis. To our knowledge, the Synechocystis sequence thus represents the first chlorophyll biosynthesis gene homolog of bchM. The complementing Synechocystis cosmid was also shown to code for a gene product that is a member of a highly conserved family of RNA binding proteins, the function of which in cyanobacteria remains undetermined.     

Effects of Iron and Oxygen on Chlorophyll Biosynthesis : I. IN VIVO OBSERVATIONS ON IRON AND OXYGEN-DEFICIENT PLANTS

Corn (Zea mays, L.), bean (Phaseolus vulgaris L.), barley (Hordeum vulgare L.), spinach (Spinacia oleracea L.), and sugarbeet (Beta vulgaris L.) grown under iron deficiency, and Potamogeton pectinatus L, and Potamogeton nodosus Poir. grown under oxygen deficiency, contained less chlorophyll than the controls, but accumulated Mg-protoporphyrin IX and/or Mg-protoporphyrin IX monomethyl ester. No significant accumulation of these intermediates was detected in the controls or in the tissue of plants stressed by S, Mg, N deficiency, or by prolonged dark treatment. Treatment of normal plant tissue with δ-aminolevulinic acid in the dark resulted in the accumulation of protochlorophyllide. If this treatment was carried out under conditions of iron or oxygen deficiency, less protochlorophyllide was formed, but a significant amount of Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester accumulated.     

Characterization of Chlamydomonas mutants defective in the H subunit of Mg-chelatase

Two chlorophyll-deficient mutants of Chlamydomonas reinhardtii, chl1 and brs-1, are light sensitive and, when grown heterotrophically in the dark, accumulate protoporphyrin IX and exhibit yellow/orange pigmentation. The lesions in both mutants were mapped to the gene (CHLH) for the plastid-localized H subunit of the heterotrimeric magnesium chelatase that catalyzes the insertion of magnesium into protoporphyrin IX. The genetic defects in the mutants could be assigned to +1 frameshift mutations in exon 9 (chl1) and exon 10 (brs-1) of the CHLH gene. In both mutants, the H subunit of magnesium chelatase was undetectable, but, as shown for chl1, the steady-state levels of the I and D subunits were unaltered in comparison to wild type. The CHLH gene exhibits marked light inducibility: levels of both the mRNA and the protein product are strongly increased when cultures are shifted from from the dark into the light, suggesting that this protein may play a crucial role in the light regulation of chlorophyll biosynthesis.     

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.     

Differential regulation of <Emphasis Type="Italic">chlorophyll a oxygenase</Emphasis> genes in rice

Chlorophyll b is synthesized from chlorophyll a by chlorophyll a oxygenase. We have identified two genes (OsCAO1 and OsCAO2) from the rice genome that are highly homologous to previously studied chlorophyll a oxygenase (CAO) genes. They are positioned in tandem, probably resulting from recent gene duplications. The proteins they encode contain two conserved functional motifs – the Rieske Fe–sulfur coordinating center and a non-heme mononuclear Fe-binding site. OsCAO1 is induced by light and is preferentially expressed in photosynthetic tissues. Its mRNA level decreases when plants are grown in the dark. In contrast, OsCAO2 mRNA levels are higher under dark conditions, and its expression is down-regulated by exposure to light. To elucidate the physiological function of the CAO genes, we have isolated knockout mutant lines tagged by T-DNA or Tos17. Mutant plants containing a T-DNA insertion in the first intron of the OsCAO1 gene have pale green leaves, indicating chlorophyll b deficiency. We have also isolated a pale green mutant with a Tos17 insertion in that OsCAO1 gene. In contrast, OsCAO2 knockout mutant leaves do not differ significantly from the wild type. These results suggest that OsCAO1 plays a major role in chlorophyll b biosynthesis, and that OsCAO2 may function in the dark.     

Accumulation of the NON‐YELLOW COLORING 1 protein of the chlorophyll cycle requires chlorophyll b in Arabidopsis thaliana

Chlorophyll a and chlorophyll b are interconverted in the chlorophyll cycle. The initial step in the conversion of chlorophyll b to chlorophyll a is catalyzed by the chlorophyll b reductases NON‐YELLOW COLORING 1 (NYC1) and NYC1‐like (NOL), which convert chlorophyll b to 7‐hydroxymethyl chlorophyll a. This step is also the first stage in the degradation of the light‐harvesting chlorophyll a/b protein complex (LHC). In this study, we examined the effect of chlorophyll b on the level of NYC1. NYC1 mRNA and NYC1 protein were in low abundance in green leaves, but their levels increased in response to dark‐induced senescence. When the level of chlorophyll b was enhanced by the introduction of a truncated chlorophyllide a oxygenase gene and the leaves were incubated in the dark, the amount of NYC1 was greatly increased compared with that of the wild type; however, the amount of NYC1 mRNA was the same as in the wild type. In contrast, NYC1 did not accumulate in the mutant without chlorophyll b, even though the NYC1 mRNA level was high after incubation in the dark. Quantification of the LHC protein showed no strong correlation between the levels of NYC1 and LHC proteins. However, the level of chlorophyll fluorescence of the dark adapted plant (F_o) was closely related to the accumulation of NYC1, suggesting that the NYC1 level is related to the energetically uncoupled LHC. These results and previous reports on the degradation of chlorophyllide a oxygenase suggest that the a feedforward and feedback network is included in chlorophyll cycle.     

Formation of Mg-Containing Chlorophyll Precursors from Protoporphyrin IX, delta-Aminolevulinic Acid, and Glutamate in Isolated, Photosynthetically Competent, Developing Chloroplasts

Intact developing chloroplasts isolated from greening cucumber (Cucumis sativus L. var Beit Alpha) cotyledons were found to contain all the enzymes necessary for the synthesis of chlorophyllide. Glutamate was converted to Mg-protoporphyrin IX (monomethyl ester) and protoclorophyllide. δ-Aminolevulinic acid and protoporphyrin IX were converted to Mg-protoporphyrin IX, Mg-protoporphyrin IX monomethyl ester, protochlorophyllide and chlorophyllide a. The conversion of δ-aminolevulinic acid or protoporphyrin IX to Mg-protoporphyrin IX (monomethyl ester) was inhibited by AMP and p-chloromercuribenzene sulfonate. Light stimulated the formation of Mg-protoporphyrin IX from all three substrates. In the case of δ-aminolevulinic acid and protoporphyrin IX, light could be replaced by exogenous ATP. In the case of glutamate, both ATP and reducing power were necessary to replace light. With all three substrates, glutamate, δ-aminolevulinic acid, and protoporphyrin IX, the stimulation of Mg-protoporphyrin IX accumulation in the light was abolished by DCMU, and this DCMU block was overcome by added ATP and reducing power.