Ali Alawady

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.

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.

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.

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.

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.

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.

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.

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.