Deficiency in riboflavin biosynthesis affects tetrapyrrole biosynthesis in etiolated Arabidopsis tissue.

Tetrapyrrole biosynthesis is controlled by multiple environmental and endogenous cues. Etiolated T-DNA insertion mutants were screened for red fluorescence as result of elevated levels of protochlorophyllide and four red fluorescent in the dark (rfd) mutants were isolated and identified. rfd3 and rfd4 belong to the group of photomorphogenic cop/det/fus mutants. rfd1 and rfd2 had genetic lesions in RIBA1 and FLU encoding the dual-functional protein GTP cyclohydrolase II/3,4-dihydroxy-2-butanone-4-phosphate synthase and a negative regulator of tetrapyrrole biosynthesis, respectively. RIBA1 catalyses the initial reaction of the metabolic pathway of riboflavin biosynthesis and rfd1 contains reduced contents of riboflavin and the flavo-coenzymes FMN and FAD. Transcriptome analysis of rfd1 revealed up-regulated genes encoding nucleus-localized factors involved in cytokinin signalling and numerous down-regulated LEA genes as well as an auxin-inducible GH3 gene. Alteration of cytokinin metabolism of rfd1was confirmed by elevated contents of active forms of cytokinin and stimulated expression of an ARR6::GUS reporter construct. An etiolated quadruple ckx (cytokinin oxidase) mutant with impaired cytokinin degradation as well as different knockout mutants for the negative AUX/IAA regulators shy2-101 (iaa3), axr2-1 (iaa7) and slr-1 (iaa14) showed also excessive protochlorophyllide accumulation. The transcript levels of CHLH and HEMA1 encoding Mg chelatase and glutamyl-tRNA reductase were increased in rfd1 and the AUX/IAA loss-of-function mutants. It is proposed that reduced riboflavin synthesis impairs the activity of the flavin-containing cytokinin oxidase, increases cytokinin contents and de-represses synthesis of 5-aminolevulinic acid of tetrapyrrole metabolism in darkness. As result of the mutant analyses, the antagonistic cytokinin and auxin signalling is required for a balanced tetrapyrrole biosynthesis in the dark.

De-regulation of abscisic acid contents causes abnormal endosperm development in the barley mutant seg8.

Grain development of the maternal effect shrunken endosperm mutant seg8 was analysed by comprehensive molecular, biochemical and histological methods. The most obvious finding was de-regulation of ABA levels, which were lower compared to wild-type during the pre-storage phase but higher during the transition from cell division/differentiation to accumulation of storage products. Ploidy levels and ABA amounts were inversely correlated in the developing endosperms of both mutant and wild-type, suggesting an influence of ABA on cell-cycle regulation. The low ABA levels found in seg8 grains between anthesis and beginning endosperm cellularization may result from a gene dosage effect in the syncytial endosperm that causes impaired transfer of ABA synthesized in vegetative tissues into filial grain parts. Increased ABA levels during the transition phase are accompanied by higher chlorophyll and carotenoid/xanthophyll contents. The data suggest a disturbed ABA-releasing biosynthetic pathway. This is indicated by up-regulation of expression of the geranylgeranyl reductase (GGR) gene, which may be induced by ABA deficiency during the pre-storage phase. Abnormal cellularization/differentiation of the developing seg8 endosperm and reduced accumulation of starch are phenotypic characteristics that reflect these disturbances. The present study did not reveal the primary gene defect causing the seg8 phenotype, but presents new insights into the maternal/filial relationships regulating barley endosperm development.

Chlorophyll-deficient mutants of Chlamydomonas reinhardtii that accumulate magnesium protoporphyrin IX.

Two Chlamydomonas reinhardtii mutants defective in CHLM encoding Mg-protoporphyrin IX methyltransferase (MgPMT) were identified. The mutants, one with a missense mutation (chlM-1) and a second mutant with a splicing defect (chlM-2), do not accumulate chlorophyll, are yellow in the dark and dim light, and their growth is inhibited at higher light intensities. They accumulate Mg-protoporphyrin IX (MgProto), the substrate of MgPMT and this may be the cause for their light sensitivity. In the dark, both mutants showed a drastic reduction in the amounts of core proteins of photosystems I and II and light-harvesting chlorophyll a/b-binding proteins. However, LHC mRNAs accumulated above wild-type levels. The accumulation of the transcripts of the LHC and other genes that were expressed at higher levels in the mutants during dark incubation was attenuated in the initial phase of light exposure. No regulatory effects of the constitutively 7- to 18-fold increased MgProto levels on gene expression were detected, supporting previous results in which MgProto and heme in Chlamydomonas were assigned roles as second messengers only in the transient activation of genes by light.

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.

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

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

HEMA RNAi silencing reveals a control mechanism of ALA biosynthesis on Mg chelatase and Fe chelatase.

Glutamyl-tRNA reductase (GluTR) is encoded by HEMA in higher plants and catalyzes in plastids the initial enzymatic step of tetrapyrrole biosynthesis eventually leading to heme and chlorophyll. GluTR activity is subjected to a complex regulation on multiple expression levels. An ethanol-inducible HEMA-RNA-interference (RNAi) gene construct was introduced into the tobacco genome to study the primary effects of low GluTR content on the tetrapyrrole biosynthetic pathway. During the first days of induced HEMA silencing the chlorophyll and heme contents were diminished in young leaves. HEMA mRNA and GluTR protein content were also strongly reduced. However, expression analyses revealed that none of the other tetrapyrrole biosynthesis genes were affected on the transcriptional level in a nine days period after HEMA inactivation. Previously generated transgenic tobacco lines with RNAi silenced expression of the glutamate 1-semialdehyde aminotransferase (GSA) gene did also not display changes of transcripts from selected genes of tetrapyrrole biosynthesis and photosynthesis. Although the transcript levels were not decreased after inactivation of HEMA and GSA-expression, enzyme activities for Mg chelatase and Fe chelatase were lower, which occurred in parallel to the loss of chlorophyll and heme content. Posttranslational modification of enzymes downstream of ALA-biosynthesis is proposed as a regulatory mechanism to adjust the flux through tetrapyrrole biosynthesis in balance to supply of ALA.

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.

Limitation of nocturnal import of ATP into Arabidopsis chloroplasts leads to photooxidative damage.

When grown in short day conditions and at low light, leaves of Arabidopsis plants with mutations in the genes encoding two plastidial ATP/ADP transporters (so-called null mutants) spontaneously develop necrotic lesions. Under these conditions, the mutants also display light-induced accumulation of H(2)O(2) and constitutive expression of genes for copper/zinc superoxide dismutase 2 and ascorbate peroxidase 1. In the light phase, null mutants accumulate high levels of phototoxic protoporphyrin IX but have only slightly reduced levels of Mg protoporphyrin IX. The physiological changes are associated with reduced magnesium-chelatase activity. Since the expression of genes encoding any of the three subunits of magnesium-chelatase is similar in wild type and null mutants, decreased enzyme activity is probably due to post-translational modification which might be due to limited availability of ATP in plastids during the night. Surprisingly, the formation of necrotic lesions was absent when null mutants were grown either in long days and low light intensity or in short days and high light intensity. We ascribe the lack of lesion phenotype to increased nocturnal ATP supply due to glycolytic degradation of starch which may lead to additional substrate-level phosphorylation in the stroma. Thus, nocturnal import of ATP into chloroplasts represents a crucial, previously unknown process that is required for controlled chlorophyll biosynthesis and for preventing photooxidative damage.

Regulation of lutein biosynthesis and prolamellar body formation in Arabidopsis.

Carotenoids are critical for photosynthetic function in chloroplasts, and are essential for the formation of the prolamellar body in the etioplasts of dark-grown (etiolated) seedlings. They are also precursors for plant hormones in both types of plastids. Lutein is one of the most abundant carotenoids found in both plastids. In this study we examine the regulation of lutein biosynthesis and investigate the effect of perturbing carotenoid biosynthesis on the formation of the lattice-like membranous structure of etioplasts, the prolamellar body (PLB). Analysis of mRNA abundance in wildtype and lutein-deficient mutants, lut2 and ccr2, in response to light transitions and herbicide treatments demonstrated that the mRNA abundance of the carotenoid isomerase (CRTISO) and epsilon-cyclase (ϵLCY) can be rate limiting steps in lutein biosynthesis. We show that accumulation of tetra-cis-lycopene and all-trans-lycopene correlates with the abundance of mRNA of several carotenoid biosynthetic genes. Herbicide treatments that inhibit carotenoid biosynthetic enzymes in wildtype and ccr2 etiolated seedlings were used to demonstrate that the loss of the PLB in ccr2 mutants is a result of perturbations in carotenoid accumulation, not indirect secondary effects, as PLB formation could be restored in ccr2 mutants treated with norflurazon.

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.

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.

Tobacco Mg protoporphyrin IX methyltransferase is involved in inverse activation of Mg porphyrin and protoheme synthesis.

Protoporphyrin, a metabolic intermediate of tetrapyrrole biosynthesis, is metabolized by Mg chelatase and ferrochelatase and is directed into the Mg-branch for chlorophyll synthesis and in the Fe-branch for protoheme synthesis respectively. Regulation of the enzyme activities at the beginning of this branchpoint ensures accurate partition of protoporphyrin, but is still not entirely understood. Transgenic tobacco plants were generated that express antisense or sense RNA for inhibited and excessive expression of Mg protoporphyrin methyltransferase (MgPMT) respectively. This enzyme accepts Mg protoporphyrin from Mg chelatase and catalyses the transfer of a methyl group to the carboxyl group of the C13-propionate side chain. Low MgPMT activity is correlated with reduced Mg chelatase activity and a low synthesis rate of 5-aminolevulinate, but with enhanced ferrochelatase activity. In contrast, high MgPMT activity leads to inverse activity profiles: high activities of Mg chelatase and for 5-aminolevulinate synthesis, but reduced activity of ferrochelatase, indicating a direct influence of MgPMT in combination with Mg chelatase on the metabolic flux of ALA and the distribution of protoporphyrin into the branched pathway. The modified enzyme activities in tetrapyrrole biosynthesis in the transgenic plants can be explained with changes of certain corresponding mRNA contents: increased 5-aminolevulinate synthesis and Mg chelatase activity correlate with enhanced transcript levels of the HemA, Gsa, and CHLH gene encoding glutamyl-tRNA reductase, glutamate-1-semialdehyde aminotransferase and a Mg chelatase subunit respectively. It is proposed that reduced and increased MgPMT activity in chloroplasts is communicated to the cytoplasm for modulating transcriptional activities of regulatory enzymes of the pathway.

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.