5-aminolevulinic Acid Dehydratase Research Articles

More indications for redox-sensitive cysteine residues of the Arabidopsis 5-aminolevulinate dehydratase.

Redox-dependent thiol-disulfide switches of cysteine residues are one of the significant posttranslational modifications of proteins to control rapidly their stability, activity, and protein interaction. Redox control also modulates the tetrapyrrole biosynthesis (TBS). Among the redox-dependent TBS enzymes, 5-aminolevulinic acid dehydratase (ALAD) was previously recognized to interact with reductants, such a thioredoxins or NADPH-dependent thioredoxin reductase C. In this report, we aim to verify the redox sensitivity of ALAD and identify the redox-reactive cysteine residues among the six cysteines of the mature protein form Arabidopsis. Based on structural modelling and comparative studies of wild-type ALAD and ALAD mutants with single and double Cys➔Ser substitutions under oxidizing and reducing conditions, we aim to predict the dimerization and oligomerisation of ALAD as well as the crucial Cys residues for disulfide bridge formation and enzyme activity. The Cys404Ser mutation led to a drastic inactivation of ALAD and redox-dependent properties of ALAD were severely impaired, when Cys71 was simultaneously mutated with Cys152 or Cys251. Cys71 is located in a flexible N-terminal arm of ALAD, which could allow intramolecular disulfide bridges with Cys residues at the surface of the remaining globule ALAD structure. As a result, we propose different roles of Cys residues for redox control, catalytic activity and Mg2+-dependent assembly.

NTRC and TRX-f Coordinately Affect the Levels of Enzymes of Chlorophyll Biosynthesis in a Light-Dependent Manner.

Redox regulation of plastid gene expression and different metabolic pathways promotes many activities of redox-sensitive proteins. We address the question of how the plastid redox state and the contributing reducing enzymes control the enzymes of tetrapyrrole biosynthesis (TBS). In higher plants, this metabolic pathway serves to produce chlorophyll and heme, among other essential end products. Because of the strictly light-dependent synthesis of chlorophyll, tight control of TBS requires a diurnal balanced supply of the precursor 5-aminolevulinic acid (ALA) to prevent the accumulation of photoreactive metabolic intermediates in darkness. We report on some TBS enzymes that accumulate in a light intensity-dependent manner, and their contents decrease under oxidizing conditions of darkness, low light conditions, or in the absence of NADPH-dependent thioredoxin reductase (NTRC) and thioredoxin f1 (TRX-f1). Analysis of single and double trxf1 and ntrc mutants revealed a decreased content of the early TBS enzymes glutamyl-tRNA reductase (GluTR) and 5-aminolevulinic acid dehydratase (ALAD) instead of an exclusive decrease in enzyme activity. This effect was dependent on light conditions and strongly attenuated after transfer to high light intensities. Thus, it is suggested that a deficiency of plastid-localized thiol-redox transmitters leads to enhanced degradation of TBS enzymes rather than being directly caused by lower catalytic activity. The effects of the proteolytic activity of the Clp protease on TBS enzymes were studied by using Clp subunit-deficient mutants. The simultaneous lack of TRX and Clp activities in double mutants confirms the Clp-induced degradation of some TBS proteins in the absence of reductive activity of TRXs. In addition, we verified previous observations that decreased chlorophyll and heme levels in ntrc could be reverted to WT levels in the ntrc/Δ2cp triple mutant. The decreased synthesis of 5-aminolevulinic acid and porphobilinogen in ntrc was completely restored in ntrc/Δ2cp and correlated with WT-like levels of GluTR, ALAD, and other TBS proteins.

Heme Synthesis Inhibition Blocks Angiogenesis via Mitochondrial Dysfunction.

SummaryThe relationship between heme metabolism and angiogenesis is poorly understood. The final synthesis of heme occurs in mitochondria, where ferrochelatase (FECH) inserts Fe2+ into protoporphyrin IX to produce proto-heme IX. We previously showed that FECH inhibition is antiangiogenic in human retinal microvascular endothelial cells (HRECs) and in animal models of ocular neovascularization. In the present study, we sought to understand the mechanism of how FECH and thus heme is involved in endothelial cell function. Mitochondria in endothelial cells had several defects in function after heme inhibition. FECH loss changed the shape and mass of mitochondria and led to significant oxidative stress. Oxidative phosphorylation and mitochondrial Complex IV were decreased in HRECs and in murine retina ex vivo after heme depletion. Supplementation with heme partially rescued phenotypes of FECH blockade. These findings provide an unexpected link between mitochondrial heme metabolism and angiogenesis.

Chlorophyll Metabolism and Gene Expression in Response to Submergence Stress and Subsequent Recovery in Perennial Ryegrass Accessions Differing in Growth Habits

Submergence-induced leaf senescence may alter chlorophyll metabolism. The objective of this study was to characterize chlorophyll biosynthesis and degradation in contrasting perennial ryegrass (Lolium perenne) in response to submergence stress and recovery. The light-green and fast-growing accession PI238938 and the darker-green and slow-growing cultivar BrightStar SLT were exposed to 0, 6 h, 1-, 3-, and 7-d of submergence stress and 1- and 5- d of de-submergence, respectively. Plant growth of PI238938 were more severely inhibited by submergence stress and recovery. Both accessions showed increased leaf malondialdehyde under stress and recovery, but reduced chlorophyll (Chl) concentrations were observed at 3- and 7-d of stress and at recovery. The reduction in Chl was more severe in BrightStar SLT at 7 d of stress. The concentration of 5-aminolevulenic acid was unaffected by stress but increased at 1d of recovery. Activities of 5-aminolevulinic acid dehydratase (ALAD) involved in Chl biosynthesis remained unchanged under stress and recovery, while the activities of Chl degrading enzymes chlorophyllase (CHL) and pheophytinase (PPH) increased at 3 d or 7 d of stress, and returned to the control level after recovery in both accessions. The downregulation of Chl-biosynthetic genes CHLI, POR, and CHLP and the upregulation of Chl-degrading genes CLH, PPH, and SGR were observed in both accessions under most of the stress periods. BrightStar SLT exhibited much lower expressions of the Chl-biosynthetic genes PBGD, CHS, and CHID under stress, while PI238938 had remarkably higher expressions of genes involved in Chl breakdown including CLH, PPH, PAO, RCCR, and SGR, and the expressions of these genes remained at a higher level at 1 d of recovery. The results indicated that submergence-induced leaf senescence and declines in Chl were associated with downregulation of more Chl-biosynthetic genes in slow-growing genotype and upregulation of more Chl-degrading genes in fast-growing genotype of perennial ryegrass.

Identification and characterization of levulinyl-CoA synthetase from Pseudomonas citronellolis, which differs phylogenetically from LvaE of Pseudomonas putida

Levulinic acid (LA) is a building block alternative to fermentable sugars derived from cellulosic biomass. Among LA catabolic processes in Pseudomonas putida KT2440, ligation of coenzyme A (CoA) to LA by levulinyl-CoA synthetase (LvaE) is known to be an initial enzymatic step in LA metabolism. To identify the genes involved in the first step of LA metabolism in Pseudomonas citronellolis LA18T, RNA-seq-based comparative transcriptome analysis was carried out for LA18T cells during growth on LA and pyruvic acid. The two most highly upregulated genes with LA exhibited amino acid sequence homologies to cation acetate symporter and 5-aminolevulinic acid dehydratase from Pseudomonas spp. Potential LA metabolic genes (lva genes) in LA18T that clustered with these two genes and were homologous to lva genes in KT2440 were identified, including lvaE2 of LA18T, which exhibited 35% identity with lvaE of KT2440. Using Escherichia coli cells with the pCold™ expression system, LvaE2 was produced and investigated for its activity toward LA. High performance liquid chromatography analysis confirmed that crude extracts of E. coli cells expressing the lvaE2 gene could convert LA to levulinyl-CoA in the presence of both HS-CoA and ATP. Phylogenetic analysis revealed that LvaE2 and LvaE formed a cluster with medium-chain fatty acid CoA synthetase, but they fell on different branches. Superimposition of LvaE2 and LvaE homology-based model structures suggested that LvaE2 had a larger tunnel for accepting fatty acid substrates than LvaE. These results indicate that LvaE2 is a novel levulinyl-CoA synthetase.

Towards Initial Indications for a Thiol-Based Redox Control of Arabidopsis 5-Aminolevulinic Acid Dehydratase.

Thiol-based redox control is one of the important posttranslational mechanisms of the tetrapyrrole biosynthesis pathway. Many enzymes of the pathway have been shown to interact with thioredoxin (TRX) and Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductase C (NTRC). We examined the redox-dependency of 5-aminolevulinic acid dehydratase (ALAD), which catalyzed the conjugation of two 5-aminolevulinic acid (ALA) molecules to porphobilinogen. ALAD interacted with TRX f, TRX m and NTRC in chloroplasts. Consequently, less ALAD protein accumulated in the trx f1, ntrc and trx f1/ntrc mutants compared to wild-type control resulting in decreased ALAD activity. In a polyacrylamide gel under non-reducing conditions, ALAD monomers turned out to be present in reduced and two oxidized forms. The reduced and oxidized forms of ALAD differed in their catalytic activity. The addition of TRX stimulated ALAD activity. From our results it was concluded that (i) deficiency of the reducing power mainly affected the in planta stability of ALAD; and (ii) the reduced form of ALAD displayed increased enzymatic activity.

Reductions in the mitochondrial ABC transporter Abcb10 affect the transcriptional profile of heme biosynthesis genes

ATP-binding cassette subfamily B member 10 (Abcb10) is a mitochondrial ATP-binding cassette (ABC) transporter that complexes with mitoferrin1 and ferrochelatase to enhance heme biosynthesis in developing red blood cells. Reductions in Abcb10 levels have been shown to reduce mitoferrin1 protein levels and iron import into mitochondria, resulting in reduced heme biosynthesis. As an ABC transporter, Abcb10 binds and hydrolyzes ATP, but its transported substrate is unknown. Here, we determined that decreases in Abcb10 did not result in protoporphyrin IX accumulation in morphant-treated zebrafish embryos or in differentiated Abcb10-specific shRNA murine Friend erythroleukemia (MEL) cells in which Abcb10 was specifically silenced with shRNA. We also found that the ATPase activity of Abcb10 is necessary for hemoglobinization in MEL cells, suggesting that the substrate transported by Abcb10 is important in mediating increased heme biosynthesis during erythroid development. Inhibition of 5-aminolevulinic acid dehydratase (EC 4.2.1.24) with succinylacetone resulted in both 5-aminolevulinic acid (ALA) accumulation in control and Abcb10-specific shRNA MEL cells, demonstrating that reductions in Abcb10 do not affect ALA export from mitochondria and indicating that Abcb10 does not transport ALA. Abcb10 silencing resulted in an alteration in the heme biosynthesis transcriptional profile due to repression by the transcriptional regulator Bach1, which could be partially rescued by overexpression of Alas2 or Gata1, providing a mechanistic explanation for why Abcb10 shRNA MEL cells exhibit reduced hemoglobinization. In conclusion, our findings rule out that Abcb10 transports ALA and indicate that Abcb10's ATP-hydrolysis activity is critical for hemoglobinization and that the substrate transported by Abcb10 provides a signal that optimizes hemoglobinization.

5-Aminolevulinic Acid Dehydratase Gene Dosage Affects Programmed Cell Death and Immunity.

Programmed cell death (PCD) is an important form to protect plants from pathogen attack. However, plants must precisely control the PCD process under microbe attacks to avoid detrimental effects. The complexity of how plants balance the defense activation and PCD requires further clarification. Lesion mimic mutants constitute an excellent material to study the crosstalk between them. Here, we identified a Gossypium hirsutum (cotton) lesion mimic mutant (Ghlmm), which exhibits necrotic leaf damage and enhanced disease resistance. Map-based cloning demonstrated that GhLMMD, encoding 5-aminolevulinic acid dehydratase and located on chromosome D5, was responsible for the phenotype. The mutant was resulted from a nonsense mutation within the coding region of GhLMMD It exhibited an overaccumulation of the 5-aminolevulinic acid, elevated levels of reactive oxygen species and salicylic acid, along with constitutive expression of pathogenesis-related genes and enhanced resistance to the Verticillium dahliae infection. Interestingly, GhLMM plays a dosage-dependent role in regulating PCD of cotton leaves and resistance to V. dahliae infection. This study provides a new strategy on the modulation of plant immunity, particularly in polyploidy plants.

The Aspergillus nidulans Velvet-interacting protein, VipA, is involved in light-stimulated heme biosynthesis.

Filamentous fungi are able to differentiate morphologically and adapt the metabolism to internal and external cues. One major regulator is the so-called velvet protein, VeA, best studied in Aspergillus nidulans. The protein interacts with several other proteins to regulate light sensing, the balance between asexual and sexual development, penicillin biosynthesis or mycotoxin production. Here, we characterized a novel VeA-interacting protein, VipA. The 334 amino acid long protein comprises a FAR1-like DNA-binding domain, known from plant transcription factors like FHY3 (Far-red elongated hypocotyl 3). VipA interacted not only with VeA, but also with the WC orthologue LreA in the nuclei and with the phytochrome FphA in the cytoplasm. Conidia and cleistothecia formation was similarly affected in a vipA-deletion strain as in an fphA mutant. However, the effect was less pronounced, suggesting a modulating and not an essential role in light sensing. In addition, VipA modulated heme biosynthesis in response to light through association with the hemB promoter, the gene encoding 5-aminolevulinic acid dehydratase. After illumination of A. nidulans mycelia with white light the intracellular heme concentration increased by 30% in comparison to a vipA-deletion mutant. Hence, VipA couples heme biosynthesis to the illumination conditions.

Reduced gene expression at the branch point of chlorophyll and heme biosynthesis in Arctic Chlorella ArM0029B

The gene expression at the branch point of chlorophyll and heme synthesis in the model microalga, Chlamydomonas reinhardtii, is different from that of higher plants. Another green alga, Arctic Chlorella, was recently isolated from Arctic sea ice and may be a promising candidate for a biofuel. To understand the chlorophyll metabolic pathway and relevant nuclear gene expression in Chlorella sp., we characterized chlorophyll-deficient mutants of the Arctic Chlorella sp. ArM0029B. First, we characterized the chlorophyll and heme biosynthetic pathways based on genes identified by bioinformatics analysis of the genome of Arctic Chlorella sp. ArM0029B. Then, we isolated and analyzed nine chlorophyll-deficient mutants that showed reduced expression of the ChlM gene, which encodes Mg-protoporphyrin methyltransferase. Expression of 5-amino levulinic acid dehydratase (encoded by ALAD) and glutamyl-tRNA reductase (encoded by HemA) was reduced in all nine independent mutants compared to wild type. These results indicated that Arctic Chlorella ArM0029B may have a regulatory mechanism of gene expression at earlier steps of the Mg-porphyrin branch that is more similar to higher plants than to the microalga C. reinhardtii. This study provides useful insight into the regulation of porphyrin precursor formation in Chlorella and related microalgae.

Modulation of biosynthesis of photosynthetic pigments and light-harvesting complex in wild-type and gun5 mutant of Arabidopsis thaliana during impaired chloroplast development.

Plants in response to different environmental cues need to modulate the expression of nuclear and chloroplast genomes that are in constant communication. To understand the signals that are responsible for inter-organellar communication, levulinic acid (LA), an inhibitor of 5-aminolevulinic acid dehydratase, was used to suppress the synthesis of pyrrole-derived tetrapyrroles chlorophylls. Although, it does not specifically inhibit carotenoid biosynthesis enzymes, LA reduced the carotenoid contents during photomorphogenesis of etiolated Arabidopsis seedlings. The expression of nuclear genes involved in carotenoid biosynthesis, i.e., geranylgeranyl diphosphate synthase, phytoene synthase, and phytoene desaturase, was downregulated in LA-treated seedlings. Similarly, the transcript abundance of nuclear genes, i.e., Lhcb1, PsbO, and RcbS, coding for chloroplastic proteins was severely attenuated in LA-treated samples. In contrast, LA treatment did not affect the transcript abundance of chalcone synthase, a marker gene for cytoplasm, and β-ATP synthase, a marker gene for mitochondria. This demonstrates the retrograde signaling from chloroplast to nucleus to suppress chloroplastic proteins during impaired chloroplast development. However, under identical conditions in LA-treated tetrapyrrole-deficient gun5 mutant, retrograde signal continued. The tetrapyrrole biosynthesis inhibitor LA suppressed formation of all tetrapyrroles both in WT and gun5. This rules out the role of tetrapyrroles as signaling molecules in WT and gun5. The removal of LA from the Arabidopsis seedlings restored the chlorophyll and carotenoid contents and expression of nuclear genes coding for chloroplastic proteins involved in chloroplast biogenesis. Therefore, LA could be used to modulate chloroplast biogenesis at a desired phase of chloroplast development.

A pair of light signaling factors FHY3 and FAR1 regulates plant immunity by modulating chlorophyll biosynthesis.

Light and chloroplast function is known to affect the plant immune response; however, the underlying mechanism remains elusive. We previously demonstrated that two light signaling factors, FAR‐RED ELONGATED HYPOCOTYL 3 (FHY3) and FAR‐RED IMPAIRED RESPONSE 1 (FAR1), regulate chlorophyll biosynthesis and seedling growth via controlling HEMB1 expression in Arabidopsis thaliana. In this study, we reveal that FHY3 and FAR1 are involved in modulating plant immunity. We showed that the fhy3 far1 double null mutant displayed high levels of reactive oxygen species and salicylic acid (SA) and increased resistance to Pseudomonas syringae pathogen infection. Microarray analysis revealed that a large proportion of pathogen‐related genes, particularly genes encoding nucleotide‐binding and leucine‐rich repeat domain resistant proteins, are highly induced in fhy3 far1. Genetic studies indicated that the defects of fhy3 far1 can be largely rescued by reducing SA signaling or blocking SA accumulation, and by overexpression of HEMB1, which encodes a 5‐aminolevulinic acid dehydratase in the chlorophyll biosynthetic pathway. Furthermore, we found that transgenic plants with reduced expression of HEMB1 exhibit a phenotype similar to fhy3 far1. Taken together, this study demonstrates an important role of FHY3 and FAR1 in regulating plant immunity, through integrating chlorophyll biosynthesis and the SA signaling pathway.

Salt‐stress induced modulation of chlorophyll biosynthesis during de‐etiolation of rice seedlings

Chlorophyll biosynthesis in plants is subjected to modulation by various environmental factors. To understand the modulation of the chlorophyll (Chl) biosynthesis during greening process by salt, 100-200 mM NaCl was applied to the roots of etiolated rice seedlings 12 h prior to the transfer to light. Application of 200 mM NaCl to rice seedlings that were grown in light for further 72 h resulted in reduced dry matter production (-58%) and Chl accumulation (-66%). Ionic imbalance due to salinity stress resulted in additional downregulation (41-45%) of seedling dry weight, Chl and carotenoid contents over and above that of similar osmotic stress induced by polyethylene glycol. Downregulation of Chl biosynthesis may be attributed to decreased activities of Chl biosynthetic pathway enzymes, i.e. 5-aminolevulinic acid (ALA) dehydratase (EC-2.4.1.24), porphobilinogen deaminase (EC-4.3.1.8), coproporphyrinogen III oxidase (EC-1.3.3.3), protoporphyrinogen IX oxidase (EC-1.3.3.4), Mg-protoporphyrin IX chelatase (EC-6.6.1.1) and protochlorophyllide oxidoreductase (EC-1.3.33.1). Reduced enzymatic activities were due to downregulation of their protein abundance and/or gene expression in salt-stressed seedlings. The extent of downregulation of ALA biosynthesis nearly matched with that of protochlorophyllide and Chl to prevent the accumulation of highly photosensitive photodynamic tetrapyrroles that generates singlet oxygen under stress conditions. Although, ALA synthesis decreased, the gene/protein expression of glutamyl-tRNA reductase (EC-1.2.1.70) increased suggesting it may play a role in acclimation to salt stress. The similar downregulation of both early and late Chl biosynthesis intermediates in salt-stressed seedlings suggests a regulatory network of genes involved in tetrapyrrole biosynthesis.

Ameliorative effects of ferulic Acid against lead acetate-induced oxidative stress, mitochondrial dysfunctions and toxicity in prepubertal rat brain.

Epidemiological evidence has shown higher susceptibility of Children to the adverse effects of lead (Pb) exposure. However, experimental studies on Pb-induced neurotoxicity in prepubertal (PP) rats are limited. The present study aimed to examine the propensity of ferulic acid (FA), a commonly occurring phenolic acid in staple foods (fruits, vegetables, cereals, coffee etc.) to abrogate Pb-induced toxicity. Initially, we characterized Pb-induced adverse effects among PP rats exposed to Pb acetate (1,000-3,000ppm in drinking water) for 5weeks in terms of locomotor phenotype, activity of 5-aminolevulinic acid dehydratase (ALAD) in the blood, blood Pb levels and oxidative stress in brain regions. Further, the ameliorative effects of oral supplements of FA (25mg/kg bw/day) were investigated in PP rats exposed to Pb (3,000ppm). Pb intoxication increased the locomotor activity and FA supplements partially reversed the phenotype, while the reduced ALAD activity was also restored. FA significantly abrogated the enhanced oxidative stress in cerebellum (Cb) and hippocampus (Hc) as evidenced in terms of ROS generation, lipid peroxidation and protein carbonyls. Further, Pb-mediated perturbations in the glutathione levels and activity of enzymic antioxidants were also markedly restored. Furthermore, the protective effect of FA was discernible in striatum in terms of reduced oxidative stress, restored cholinergic activity and dopamine levels. Interestingly, reduced activity levels of mitochondrial complex I in Cb and enhanced levels in Hc among Pb-intoxicated rats were ameliorated by FA supplements. FA also decreased the number of damaged cells in cornu ammonis area CA1 and dentate gyrus as reflected by the histoarchitecture of Hc among Pb intoxicated rats. Collectively, our findings in the PP model allow us to hypothesize that ingestion of common phenolics such as FA may significantly alleviate the neurotoxic effects of Pb which may be largely attributed to its ability to abrogate oxidative stress.

Inhibition of 5-aminolevulinic acid dehydratase by mercury in excised greening maize leaf segments

Mercury (Hg), a potent metallic toxicant, is known for having inhibitory effect on chlorophyll biosynthesis. Invivo supply of HgCl(2) inhibited 5-aminolevulinic acid dehydratase (ALAD, EC 4.2.1.24) activity in excised greening maize (Zea mays) leaf segments. The inhibition caused by Hg was alleviated by addition of KNO(3). Amongst the nutrients and metabolites tested, NH(4)Cl and sucrose increased the inhibitory effect of Hg on enzyme activity, while glutamine and glutathione decreased it. The inhibitors, levulinic acid and 5,5' dithio bis 2-nitrobenzoic acid, also reduced the % inhibition of enzyme activity caused by Hg supply. Invitro inclusion of Hg during assay of the enzyme preparations obtained from the tissue treated without Hg (-Hg enzyme) and with Hg (+Hg enzyme) caused the inhibition of-Hg enzyme but activation of+Hg enzyme. Almost similar trend was observed for the invitro inclusion of Hg in the presence of levulinic acid. It is suggested that two forms of enzyme exist in Hg-treated tissue, i.e. the usual Mg dependent form and an unusual Hg modified form. Kinetic studies for the two enzymes,-Hg enzyme and+Hg enzyme, involving the effect of varying concentrations of δ-aminolevulinic acid yielded distinct apparent K(m) and apparent V(max) values being 532μM and 118unitsg(-1)fr.wt., respectively, for-Hg enzyme and 347μM and 52unitsg(-1)fr.wt., respectively, for+Hg enzyme indicating that+Hg enzyme has higher affinity for δ-aminolevulinic acid but lower activity as compared to the-Hg enzyme.

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.

Transposase-Derived Proteins FHY3/FAR1 Interact with PHYTOCHROME-INTERACTING FACTOR1 to Regulate Chlorophyll Biosynthesis by Modulating HEMB1 during Deetiolation in Arabidopsis

Successful chlorophyll biosynthesis during initial light exposure is critical for plant survival and growth, as excess accumulation of chlorophyll precursors in darkness can cause photooxidative damage to cells. Therefore, efficient mechanisms have evolved to precisely regulate chlorophyll biosynthesis in plants. Here, we identify FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and FAR-RED IMPAIRED RESPONSE1 (FAR1), two transposase-derived transcription factors, as positive regulators of chlorophyll biosynthesis in Arabidopsis thaliana. We show that null mutations in FHY3 and FAR1 cause reduced protochlorophyllide (a precursor of chlorophyll) levels in darkness and less photobleaching in the light. We find that FHY3 directly binds to the promoter and activates expression of HEMB1, which encodes 5-aminolevulinic acid dehydratase in the chlorophyll biosynthetic pathway. We reveal that PHYTOCHROME-INTERACTING FACTOR1 physically interacts with the DNA binding domain of FHY3, thereby partly repressing FHY3/FAR1-activated HEMB1 expression. Strikingly, FHY3 expression is upregulated by white light. In addition, our genetic data indicate that overexpression, severe reduction, or lack of HEMB1 impairs plant growth and development. Together, our findings reveal a crucial role of FHY3/FAR1 in regulating chlorophyll biosynthesis, thus uncovering a new layer of regulation by which light promotes plant dark-light transition in early seedling development.

ZIP8 Zinc Transporter: Indispensable Role for Both Multiple-Organ Organogenesis and Hematopoiesis In Utero

Previously this laboratory characterized Slc39a8-encoded ZIP8 as a Zn2+/(HCO3 –)2 symporter; yet, the overall physiological importance of ZIP8 at the whole-organism level remains unclear. Herein we describe the phenotype of the hypomorphic Slc39a8(neo/neo) mouse which has retained the neomycin-resistance gene in intron 3, hence causing significantly decreased ZIP8 mRNA and protein levels in embryo, fetus, placenta, yolk sac, and several tissues of neonates. The Slc39a8(neo) allele is associated with diminished zinc and iron uptake in mouse fetal fibroblast and liver-derived cultures; consequently, Slc39a8(neo/neo) newborns exhibit diminished zinc and iron levels in several tissues. Slc39a8(neo/neo) homozygotes from gestational day(GD)-11.5 onward are pale, growth-stunted, and die between GD18.5 and 48 h postnatally. Defects include: severely hypoplastic spleen; hypoplasia of liver, kidney, lung, and lower limbs. Histologically, Slc39a8(neo/neo) neonates show decreased numbers of hematopoietic islands in yolk sac and liver. Low hemoglobin, hematocrit, red cell count, serum iron, and total iron-binding capacity confirmed severe anemia. Flow cytometry of fetal liver cells revealed the erythroid series strikingly affected in the hypomorph. Zinc-dependent 5-aminolevulinic acid dehydratase, required for heme synthesis, was not different between Slc39a8(+/+) and Slc39a8(neo/neo) offspring. To demonstrate further that the mouse phenotype is due to ZIP8 deficiency, we bred Slc39a8(+/neo) with BAC-transgenic BTZIP8-3 line (carrying three extra copies of the Slc39a8 allele); this cross generated viable Slc39a8(neo/neo)_BTZIP8-3(+/+) pups showing none of the above-mentioned congenital defects–proving Slc39a8(neo/neo) causes the described phenotype. Our study demonstrates that ZIP8-mediated zinc transport plays an unappreciated critical role during in utero and neonatal growth, organ morphogenesis, and hematopoiesis.

Heme Oxygenase 1 Plays An Unexpected Role During Erythroid Differentiation

Abstract 344Red blood cells (RBC) are produced at a rate of 2.3 × 106 cells per second by a dynamic and exquisitely regulated process known as erythropoiesis. During this development, RBC precursors synthesize the highest amounts of total organismal heme (75–80%), which is a complex of iron with protoporphyrin IX. Heme is essential for the function of all aerobic cells, but if left unbound to protein, it can promote free radical formation and peroxidation reactions leading to cell damage and tissue injury. Therefore, in order to prevent the accumulation of ‘free’ heme, it is imperative that cells maintain a balance of heme biosynthesis and catabolism. Physiologically, the only enzyme capable of degrading heme are heme oxyganase 1 & 2 (HO). Red blood cells contain the majority of heme destined for catabolism; this process takes place in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Heme oxygenase, in particular its heme-inducible isoform HO1, has been extensively studied in hepatocytes and many other non-erythroid cells. In contrast, virtually nothing is known about the expression of HO1 in developing RBC. Likewise, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. Using primary erythroid cells isolated from mouse fetal livers (FL), we have shown that HO1 mRNA and protein are expressed in undifferenetiated FL cells and that its levels, somewhat surprisingly, increase during erythropoietin-induced erythroid differentiation. This increase in HO1 can be prevented by succinylacetone (SA), an inhibitor of heme synthesis that blocks 5-aminolevulinic acid dehydratase, the second enzyme in the heme biosynthesis pathway. Moreover, we have found that down-regulation of HO1 via siRNA increases globin protein levels in DMSO-induced murine erythroleukemic (MEL) cells. Similarly, compared to wild type mice, FL cells isolated from HO1 knockout mice (FL/HO1−/−) exhibited increased globin and transferrin receptor levels and a decrease in ferritin levels when induced for differentiation with erythropoietin. Following induction, compared to wild type cells, FL/HO1−/− cells showed increased iron uptake and its incorporation into heme. We therefore conclude that the normal hemoglobinization rate appears to require HO1. On the other hand, MEL cells engineered to overexpress HO1 displayed reduced globin mRNA and protein levels when induced to differentiate. This finding suggests that HO1 could play a role in some pathophysiological conditions such as unbalanced globin synthesis in thalassemias. Disclosures:No relevant conflicts of interest to declare.

Comprehensive Analysis of 5-Aminolevulinic Acid Dehydrogenase (ALAD) Variants and Renal Cell Carcinoma Risk among Individuals Exposed to Lead

BackgroundEpidemiologic studies are reporting associations between lead exposure and human cancers. A polymorphism in the 5-aminolevulinic acid dehydratase (ALAD) gene affects lead toxicokinetics and may modify the adverse effects of lead.MethodsThe objective of this study was to evaluate single-nucleotide polymorphisms (SNPs) tagging the ALAD region among renal cancer cases and controls to determine whether genetic variation alters the relationship between lead and renal cancer. Occupational exposure to lead and risk of cancer was examined in a case-control study of renal cell carcinoma (RCC). Comprehensive analysis of variation across the ALAD gene was assessed using a tagging SNP approach among 987 cases and 1298 controls. Occupational lead exposure was estimated using questionnaire-based exposure assessment and expert review. Odds ratios (OR) and 95% confidence intervals (CI) were calculated using logistic regression.ResultsThe adjusted risk associated with the ALAD variant rs8177796CT/TT was increased (OR = 1.35, 95%CI = 1.05–1.73, p-value = 0.02) when compared to the major allele, regardless of lead exposure. Joint effects of lead and ALAD rs2761016 suggest an increased RCC risk for the homozygous wild-type and heterozygous alleles (GGOR = 2.68, 95%CI = 1.17–6.12, p = 0.01; GAOR = 1.79, 95%CI = 1.06–3.04 with an interaction approaching significance (pint = 0.06).. No significant modification in RCC risk was observed for the functional variant rs1800435(K68N). Haplotype analysis identified a region associated with risk supporting tagging SNP results.ConclusionA common genetic variation in ALAD may alter the risk of RCC overall, and among individuals occupationally exposed to lead. Further work in larger exposed populations is warranted to determine if ALAD modifies RCC risk associated with lead exposure.