2-alkenal Reductase Research Articles

Cold stress-induced changes in metabolism of carbonyl compounds and membrane fatty acid composition in chickpea

In this study, changes in membrane fatty acid (FA) composition and damage indices contents as well as the transcript patterns of carbonyl-detoxifying genes were evaluated in two chickpea (Cicer arietinum L.) genotypes, cold-tolerant Sel96th11439 and cold-sensitive ILC533 under cold stress (CS; 4 °C). During CS, H2O2 and malondialdehyde (MDA) contents increased (by 47% and 57%, respectively) in the sensitive genotype, while these contents remained unchanged in the tolerant genotype. In tolerant plants, higher content of linoleic, linolenic, unsaturated FAs (UFAs), total FAs and double bond index (DBI) (by 23, 21, 19, 17 and 9%, respectively) was observed at 6 days after stress (DAS) compared to sensitive plants, which, along with alterations of the damage indices, indicate their enhanced tolerance to CS. Compared with the sensitive genotype, less lipoxygenase (LOX) activity (by 59%) in the tolerant genotype was accompanied by decreased MDA and increased levels of UFAs and DBI during CS, particularly at 6 DAS. Upregulation of aldehyde dehydrogenase and aldo-keto reductase genes (by 9- and 10-fold, respectively) at 1 DAS, along with the enhanced transcript levels of aldehyde reductase and 2-alkenal reductase (by 3- and 14.7-fold, respectively) at 6 DAS were accompanied by increased UFAs and reduced MDA contents in the tolerant genotype. Overall, the results suggest that cold tolerance in chickpea was partly associated with regulation of membrane FA compositions and the potential metabolic networks involved in synthesis and degradation of carbonyl compounds.

DNA and coding/non-coding RNA methylation analysis provide insights into tomato fruit ripening.

Ripening is the last, irreversible developmental stage during which fruit become palatable, thus promoting seed dispersal by frugivory. In Alisa Craig fruit, mRNAs with increasing m5C levels, such as STPK and WRKY 40, were identified as being involved in response to biotic and abiotic stresses. Furthermore, two mRNAs involved in cell wall metabolism, PG and EXP-B1, also presented increased m5C levels. In the Nr mutant, several m5C-modified mRNAs involved in fruit ripening, including those encoding WRKY and MADS-box proteins, were found. Targets of long non-coding RNAs and circular RNAs with different m5C sites were also found; these targets included 2-alkenal reductase, soluble starch synthase 1, WRKY, MADS-box, and F-box/ketch-repeat protein SKIP11. A combined analysis of changes in 5mC methylation and mRNA revealed many differentially expressed genes with differentially methylated regions encoding transcription factors and key enzymes related to ethylene biosynthesis and signal transduction; these included ERF084, EIN3, AP2/ERF, ACO5, ACS7, EIN3/4, EBF1, MADS-box, AP2/ERF, and ETR1. Taken together, our findings contribute to the global understanding of the mechanisms underlying fruit ripening, thereby providing new information for both fruit and post-harvest behavior.

Identification of Gene Biomarkers for Tigilanol Tiglate Content in Fontainea picrosperma.

Tigilanol tiglate (EBC-46) is a small-molecule natural product under development for the treatment of cancers in humans and companion animals. The drug is currently produced by purification from the Australian rainforest tree Fontainea picrosperma (Euphorbiaceae). As part of a selective-breeding program to increase EBC-46 yield from F. picrosperma plantations, we investigated potential gene biomarkers associated with biosynthesis of EBC-46. Initially, we identified individual plants that were either high (>0.039%) or low EBC-46 (<0.008%) producers, then assessed their differentially expressed genes within the leaves and roots of these two groups by quantitative RNA sequencing. Compared to low EBC-46 producers, high-EBC-46-producing plants were found to have 145 upregulated genes and 101 downregulated genes in leaves and 53 upregulated genes and 82 downregulated genes in roots. Most of these genes were functionally associated with defence, transport, and biosynthesis. Genes identified as expressed exclusively in either the high or low EBC-46-producing plants were further validated by quantitative PCR, showing that cytochrome P450 94C1 in leaves and early response dehydration 7.1 and 2-alkenal reductase in roots were consistently and significantly upregulated in high-EBC-46 producers. In summary, this study has identified biomarker genes that may be used in the selective breeding of F. picrosperma.

Molecular and Physiological Responses of Citrus sinensis Leaves to Long-Term Low pH Revealed by RNA-Seq Integrated with Targeted Metabolomics.

Low pH-induced alterations in gene expression profiles and organic acids (OA) and free amino acid (FAA) abundances were investigated in sweet orange [Citrus sinensis (L.) Osbeck cv. Xuegan] leaves. We identified 503 downregulated and 349 upregulated genes in low pH-treated leaves. Further analysis indicated that low pH impaired light reaction and carbon fixation in photosynthetic organisms, thereby lowering photosynthesis in leaves. Low pH reduced carbon and carbohydrate metabolisms, OA biosynthesis and ATP production in leaves. Low pH downregulated the biosynthesis of nitrogen compounds, proteins, and FAAs in leaves, which might be conducive to maintaining energy homeostasis during ATP deprivation. Low pH-treated leaves displayed some adaptive responses to phosphate starvation, including phosphate recycling, lipid remodeling, and phosphate transport, thus enhancing leaf acid-tolerance. Low pH upregulated the expression of some reactive oxygen species (ROS) and aldehyde detoxifying enzyme (peroxidase and superoxidase) genes and the concentrations of some antioxidants (L-tryptophan, L-proline, nicotinic acid, pantothenic acid, and pyroglutamic acid), but it impaired the pentose phosphate pathway and VE and secondary metabolite biosynthesis and downregulated the expression of some ROS and aldehyde detoxifying enzyme (ascorbate peroxidase, aldo-keto reductase, and 2-alkenal reductase) genes and the concentrations of some antioxidants (pyridoxine and γ-aminobutyric acid), thus disturbing the balance between production and detoxification of ROS and aldehydes and causing oxidative damage to leaves.

Associated SNPs, Heritabilities, Trait Correlations, and Genomic Breeding Values for Resistance in Snap Beans (Phaseolus vulgaris L.) to Root Rot Caused by Fusarium solani (Mart.) f. sp. phaseoli (Burkholder)

Root rot is a major constraint to snap bean (Phaseolus vulgaris) production in the United States and around the world. Genetic resistance is needed to effectively control root rot disease because cultural control methods are ineffective, and the pathogen will be present at the end of one season of production on previously clean land. A diversity panel of 149 snap bean pure lines was evaluated for resistance to Fusarium root rot in Oregon. Morphological traits potentially associated with root rot resistance, such as aboveground biomass, adventitious roots, taproot diameter, basal root diameter, deepest root angle, shallowest root angle, root angle average, root angle difference, and root angle geometric mean were evaluated and correlated to disease severity. A genome wide association study (GWAS) using the Fixed and random model Circulating Probability Unification (FarmCPU) statistical method, identified five associated single nucleotide polymorphisms (SNPs) for disease severity and two SNPs for biomass. The SNPs were found on Pv03, Pv07, Pv08, Pv10, and Pv11. One candidate gene for disease reaction near a SNP on Pv03 codes for a peroxidase, and two candidates associated with biomass SNPs were a 2-alkenal reductase gene cluster on Pv10 and a Pentatricopeptide repeat domain on Pv11. Bean lines utilized in the study were ranked by genomic estimated breeding values (GEBV) for disease severity, biomass, and the root architecture traits, and the observed and predicted values had high to moderate correlations. Cross validation of genomic predictions showed slightly lower correlational accuracy. Bean lines with the highest GEBV were among the most resistant, but did not necessarily rank at the very top numerically. This study provides information on the relationship of root architecture traits to root rot disease reaction. Snap bean lines with genetic merit for genomic selection were identified and may be utilized in future breeding efforts.

Toxicity of reactive carbonyl compounds to plants

In plants, environmental stresses result in oxidative stress, lipid peroxidation and the generation of reactive carbonyl aldehydes. Reactive carbonyl aldehydes are downstream products of reactive oxygen species which can be described as critical cell-damaging agents in plants under various environmental stresses. In this paper toxicity of reactive carbonyl aldehydes and its generation under stress conditions are discussed. Moreover, involvement of reactive carbonyl aldehydes in stress- induced damage to plants is demonstrated. Toxic effect of reactive aldehydes such as acrolein, malondialdehyde and crotonaldehyde in plants under different stresses and their high electrophilicity is also discussed. Increases in malondialdehyde was demonstrated in UV-C stressed plants as the result of carbonyl modified proteins. A malondialdehyde is one of the widely shown aldehyde, which can be demonstrated as an indicator of reactive oxygen species. Malondialdehyde isomerized to 3-hydroxyacrolein whereas it can be described as a dialdehyde. The article considers detrimental actions of reactive carbonyl aldehydes and their chemical properties as well as detoxification of reactive carbonyl aldehydes by multiple enzymes such as aldehyde dehydrogenase, aldehyde reductase, aldo-keto reductase and 2-alkenal reductase.

Up-regulated 2-alkenal reductase expression improves low-nitrogen tolerance in maize by alleviating oxidative stress.

In plants, cellular lipid peroxidation is enhanced under low nitrogen (LN) stress; this increases the lipid-derived reactive carbonyl species (RCS) levels. The cellular toxicity of RCS can be reduced by various RCS-scavenging enzymes. However, the roles of these enzymes in alleviating oxidative stress and improving nutrient use efficiency (NUE) under nutrient stress remain unknown. Here, we overexpressed maize endogenous NADPH-dependent 2-alkenal reductase (ZmAER) in maize; it significantly increased the tolerance of transgenic plants (OX-AER) to LN stress. Under LN condition, the biomass, nitrogen accumulation, NUE, and leaf photosynthesis of the OX-AER plants were significantly higher than those of the wild-type (WT) plants. The leaf and root malondialdehyde and H2 O2 levels in the transgenic plants were significantly lower than those in WT. The expression of antioxidant enzyme-related genes ZmCAT3, ZmPOD5 and ZmPOD13 was significantly higher in the transgenic lines than in WT. Under LN stress, the nitrate reductase activity in the OX-AER leaves was significantly increased compared with that in the WT leaves. Furthermore, under LN stress, ZmNRT1.1 and ZmNRT2.5 expression was upregulated in the OX-AER plants compared with that in WT. Overall, up-regulated ZmAER expression could enhance maize's tolerance to LN stress by alleviating oxidative stress and improve NUE.

Reactive Carbonyl Species Mediate Methyl Jasmonate-Induced Stomatal Closure.

Production of reactive oxygen species (ROS) is a key signal event for methyl jasmonate (MeJA)- and abscisic acid (ABA)-induced stomatal closure. We recently showed that reactive carbonyl species (RCS) stimulates stomatal closure as an intermediate downstream of hydrogen peroxide (H2O2) production in the ABA signaling pathway in guard cells of Nicotiana tabacum and Arabidopsis thaliana. In this study, we examined whether RCS functions as an intermediate downstream of H2O2 production in MeJA signaling in guard cells using transgenic tobacco plants overexpressing A. thaliana 2-alkenal reductase (n-alkanal + NAD(P)+ ⇌ 2-alkenal + NAD(P)H + H+) (AER-OE tobacco) and Arabidopsis plants. The stomatal closure induced by MeJA was impaired in the AER-OE tobacco and was inhibited by RCS scavengers, carnosine and pyridoxamine, in the wild-type (WT) tobacco plants and Arabidopsis plants. Application of MeJA significantly induced the accumulation of RCS, including acrolein and 4-hydroxy-(E)-2-nonenal, in the WT tobacco but not in the AER-OE plants. Application of MeJA induced H2O2 production in the WT tobacco and the AER-OE plants and the H2O2 production was not inhibited by the RCS scavengers. These results suggest that RCS functions as an intermediate downstream of ROS production in MeJA signaling and in ABA signaling in guard cells.

Promoter deletion analysis reveals root-specific expression of the alkenal reductase gene (OsAER1) in Oryza sativa.

Root-specific promoters are useful in plant genetic engineering, primarily to improve water and nutrient absorption. The aim of this study was to clone and characterise the promoter of the Oryza sativa L. alkenal reductase (OsAER1) gene encoding 2-alkenal reductase, an NADPH-dependent oxidoreductase. Expression analysis using quantitative real-time PCR confirmed the root-specific expression of the OsAER1 gene. Subsequently, a 3082-bp fragment of the OsAER1 promoter was isolated from a local Indonesian rice cultivar, Awan Kuning. Sequencing and further nucleotide sequence analysis of the 3082-bp promoter fragment (PA-5) revealed the presence of at least 10 root-specific cis-regulatory elements putatively responsible for OsAER1 root-specific expression. Using the 3082-bp promoter fragment to drive the expression of the GUS reporter transgene confirmed that the OsAER1 promoter is root-specific. Further, the analysis indicated that OsAER1 promoter activity was absent in leaves, petioles and shoots during sprouting, vegetative, booting and generative stages of rice development. In contrast, the promoter activity was present in anthers and aleurone layers of immature seeds 7-20 days after anthesis. Moreover, there was no promoter activity observed in the aleurone layers of mature seeds. The OsAER1 promoter activity is induced by Al-toxicity, NaCl and submergence stresses, indicating the OsAER1 promoter activity is induced by those stresses. Exogenous treatments of transgenic plants carrying the PA-5 promoter construct with abscisic acid and indoleacetic acid also induced expression of the GUS reporter transgene, indicating the role of plant growth regulators in controlling OsAER1 promoter activity. Promoter deletion analysis was conducted to identify the cis-acting elements of the promoter responsible for controlling root-specific expression. The GUS reporter gene was fused with various deletion fragments of the OsAER1 promoter and the resulting constructs were transformed in rice plants to generate transgenic plants. The results of this analysis indicated that cis-acting elements controlling root-specific expression are located between -1562 to -1026bp of the OsAER1 CDS. Here we discusses the results of the conducted analyses, the possible role of OsAER1 in rice growth and development, possible contributions and the potential usage of these findings in future plant research.

Monoecious Mutant Reveals New Insights into Male and Female Inflorescence Development in the Chinese Bayberry (Morella rubra)

To identify the dynamic differences in transcriptome and gene expression in chinese bayberry flowers of different sex types, the female ‘Fugong-1’ (WT) and its monoecious mutant (MT) were used as experimental materials. Using Illumina HiSeq™ 2500, flowers at the inflorescence germination, inflorescence elongation, and initial flowering stages were sequenced by RNA-seq technology and 6 libraries were obtained. After sequence assemble, 84,945 unigenes greater than 200 bp were found and the total length was 71.66 Mb. Transcriptomic expression analysis of the six libraries indicated that there was only 297 genes showed different expression at the inflorescence germination stage between MT and WT, the difference of which was the minimum. At the elongation stage and the initial flowering stage, such numbers increased to 787 and 2722, respectively. Gene ontology (GO) functional enrichment analysis revealed that the enriched differentially expressed genes (DEGs) included those related with transcription in DNA-templated (GO: 0006355), pollen exine formation (GO: 0010584), plasma membrane (GO: 0005886), sequence-specific DNA binding transcription factor activity (GO: 0003700), and 2-alkenal reductase [NAD(P)] activity (GO: 0032440) GO categories, etc. Among these processes, pollen exine formation plays an important role in pollen cell wall synthesis and sporopollen participates in the biosynthesis of sporopollenin. High expression levels of these related genes were closely related to the MT’s male flower traits during the initial flowering stage, which resulted in the male characteristics in MT flower. This study provides important foundation for further mining important genes and regulating factors controlling the sex differentiation of flowers in chinese bayberry.

Reactive Carbonyl Species Mediate ABA Signaling in Guard Cells.

Drought is responsible for a massive reduction in crop yields. In response to drought, plants synthesize the hormone ABA, which induces stomatal closure, thus reducing water loss. In guard cells, ABA triggers production of reactive oxygen species (ROS), which is mediated by NAD(P)H oxidases. The production of ROS is a key factor for ABA-induced stomatal closure, but it remains to be clarified how the production of ROS is transduced into downstream signaling components in guard cells. We investigated roles of reactive carbonyl species (RCS) in ABA-induced stomatal closure using transgenic tobacco (Nicotiana tabacum) overexpressing Arabidopsis 2-alkenal reductase (AER-OE), which scavenges RCS. ABA and hydrogen peroxide (H2O2) induced accumulation of RCS including acrolein and 4-hydroxy-(E)-2-nonenal in wild-type tobacco but not in AER-OE. Stomatal closure and RCS accumulation in response to ABA and H2O2 were inhibited in AER-OE unlike in the wild type, while ABA-induced H2O2 production in guard cells was observed in AER-OE as well as in the wild type. Moreover, ABA inhibited inward-rectifying K+ channels in wild-type guard cells but not in AER-OE guard cells. These results suggest that RCS is involved in ABA-induced stomatal closure and functions downstream of H2O2 production in the ABA signaling pathway in guard cells.

Lipid Peroxide-Derived Short-Chain Carbonyls Mediate Hydrogen Peroxide-Induced and Salt-Induced Programmed Cell Death in Plants.

Lipid peroxide-derived toxic carbonyl compounds (oxylipin carbonyls), produced downstream of reactive oxygen species (ROS), were recently revealed to mediate abiotic stress-induced damage of plants. Here, we investigated how oxylipin carbonyls cause cell death. When tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells were exposed to hydrogen peroxide, several species of short-chain oxylipin carbonyls [i.e. 4-hydroxy-(E)-2-nonenal and acrolein] accumulated and the cells underwent programmed cell death (PCD), as judged based on DNA fragmentation, an increase in terminal deoxynucleotidyl transferase dUTP nick end labeling-positive nuclei, and cytoplasm retraction. These oxylipin carbonyls caused PCD in BY-2 cells and roots of tobacco and Arabidopsis (Arabidopsis thaliana). To test the possibility that oxylipin carbonyls mediate an oxidative signal to cause PCD, we performed pharmacological and genetic experiments. Carnosine and hydralazine, having distinct chemistry for scavenging carbonyls, significantly suppressed the increase in oxylipin carbonyls and blocked PCD in BY-2 cells and Arabidopsis roots, but they did not affect the levels of ROS and lipid peroxides. A transgenic tobacco line that overproduces 2-alkenal reductase, an Arabidopsis enzyme to detoxify α,β-unsaturated carbonyls, suffered less PCD in root epidermis after hydrogen peroxide or salt treatment than did the wild type, whereas the ROS level increases due to the stress treatments were not different between the lines. From these results, we conclude that oxylipin carbonyls are involved in the PCD process in oxidatively stressed cells. Our comparison of the ability of distinct carbonyls to induce PCD in BY-2 cells revealed that acrolein and 4-hydroxy-(E)-2-nonenal are the most potent carbonyls. The physiological relevance and possible mechanisms of the carbonyl-induced PCD are discussed.

Isolation and characterization of AaAER, a novel double bond reductase from Artemisia annua

We isolated a 2-alkenal reductase (AaAER) gene from Artemisia annua L. cDNA library through homologous cloning strategy. Enzymatic properties of AaAER, including substrate selectivity, enzyme kinetics, and key factors affecting enzyme activity, were characterized in vitro. We found that AaAER mainly functions in catalyzing the reduction of adjacent straight chain “C = C” double bond in the carbonyl group. This suggests the possibility of its participation in downstream events of the HPL (hydroperoxide lyase) pathway and contribution to cell detoxification in plants. Although, AaAER shares some substrates with AaDbr 1 and AaDbr 2, which are involved in artemisinin biosynthesis, AaAER was unable to reduce the adjacent double bond of carbonyl group in artemisinic aldehyde. We also analyzed the possible causes for highly homologous reductases with different types of substrates and non-homologous enzymes sharing the same substrate. The identification of a novel double bond reductase in A. annua provides an opportunity to explore the mechanism underlying the detoxification of aldehyde ketone molecules, which are generated by HPL downstream pathway and the redox process in the metabolic pathway of artemisinin synthesis.

Why don't plants have diabetes? Systems for scavenging reactive carbonyls in photosynthetic organisms

In the present paper, we review the toxicity of sugar- and lipid-derived RCs (reactive carbonyls) and the RC-scavenging systems observed in photosynthetic organisms. Similar to heterotrophs, photosynthetic organisms are exposed to the danger of RCs produced in sugar metabolism during both respiration and photosynthesis. RCs such as methylglyoxal and acrolein have toxic effects on the photosynthetic activity of higher plants and cyanobacteria. These toxic effects are assumed to occur uniquely in photosynthetic organisms, suggesting that RC-scavenging systems are essential for their survival. The aldo-keto reductase and the glyoxalase systems mainly scavenge sugar-derived RCs in higher plants and cyanobacteria. 2-Alkenal reductase and alkenal/alkenone reductase catalyse the reduction of lipid-derived RCs in higher plants. In cyanobacteria, medium-chain dehydrogenases/reductases are the main scavengers of lipid-derived RCs.

Accumulation of lipid peroxide-derived, toxic .ALPHA.,.BETA.-unsaturated aldehydes (E)-2-pentenal, acrolein and (E)-2-hexenal in leaves under photoinhibitory illumination

Lipid peroxide-derived α,β-unsaturated aldehydes (2-alkenals) are potent toxins that inhibit enzymes in the Calvin cycle and in the mitochondrial photorespiratory pathway. Production of 2-alkenals in illuminated leaves and their participation in cellular damages have been suggested from the observation that the transgenic tobaccos overexpressing 2-alkenal reductase (AER), a 2-alkenal-detoxifying enzyme, showed tolerance to strong light. In order to identify the involved 2-alkenal species, we here analyzed the leaf aldehyde compositions in the AER-overproducing tobaccos and wild type (SR1), and compared their changes under photoinhibitory light. Aldehydes were extracted from the leaves at an early stage of photoinhibition, derivatized with 2,4-dinitrophenylhydrazine and analyzed on reversed-phase HPLC. In dark-adapted leaves of these tobacco lines, more than 40 carbonyl species were found, of which 17 were identified. In SR1 leaves, 2-alkenals such as (E)-2-pentenal, acrolein, and (E)-2-hexenal were increased by 70–290% after 30 min-illumination. In the leaves of AER-tobaccos, light-dependent increase of these 2-alkenals was apparently lower. Thus the production of highly reactive 2-alkenals in leaves was enhanced under photooxidative stress. The tolerance to strong light due to the overexpression of AER can be explained by the scavenging of these species.

The involvement of lipid peroxide-derived aldehydes in aluminum toxicity of tobacco roots.

Oxidative injury of the root elongation zone is a primary event in aluminum (Al) toxicity in plants, but the injuring species remain unidentified. We verified the hypothesis that lipid peroxide-derived aldehydes, especially highly electrophilic alpha,beta-unsaturated aldehydes (2-alkenals), participate in Al toxicity. Transgenic tobacco (Nicotiana tabacum) overexpressing Arabidopsis (Arabidopsis thaliana) 2-alkenal reductase (AER-OE plants), wild-type SR1, and an empty vector-transformed control line (SR-Vec) were exposed to AlCl(3) on their roots. Compared with the two controls, AER-OE plants suffered less retardation of root elongation under AlCl(3) treatment and showed more rapid regrowth of roots upon Al removal. Under AlCl(3) treatment, the roots of AER-OE plants accumulated Al and H(2)O(2) to the same levels as did the sensitive controls, while they accumulated lower levels of aldehydes and suffered less cell death than SR1 and SR-Vec roots. In SR1 roots, AlCl(3) treatment markedly increased the contents of the highly reactive 2-alkenals acrolein, 4-hydroxy-(E)-2-hexenal, and 4-hydroxy-(E)-2-nonenal and other aldehydes such as malondialdehyde and formaldehyde. In AER-OE roots, accumulation of these aldehydes was significantly less. Growth of the roots exposed to 4-hydroxy-(E)-2-nonenal and (E)-2-hexenal were retarded more in SR1 than in AER-OE plants. Thus, the lipid peroxide-derived aldehydes, formed downstream of reactive oxygen species, injured root cells directly. Their suppression by AER provides a new defense mechanism against Al toxicity.

Molecular cloning and characterization of Dbr1, a 2-alkenal reductase from Artemisia annuaThe nucleotide sequence reported in this article has been deposited in the GenBank database under accession No. FJ750460.This paper is one of a selection of papers published in a Special Issue from the National Research Council of Canada – Plant Biotechnology Institute.

The molecular genetics of carbon–carbon double bond reduction in the plant Artemisia annua L. was studied. Expressed sequence tags from this plant were investigated for sequences with similarity to known double-bond reductases. This resulted in the isolation of a cDNA, corresponding to the gene A. annua Dbr1 (Double bond reductase1), encoding a member of the medium chain dehydrogenase/reductase protein superfamily with sequence similarity to tobacco allyl alcohol dehydrogenase. Recombinant A. annua Dbr1 protein was purified from Escherischia coli and shown to catalyze the reduction of the carbon–carbon double bond of 2-alkenals. This activity included the reduction of the double bond at C11–C13 in the artemisinin precursor artemisinic aldehyde, albeit with unnatural stereochemistry. The substrate specificity, product stereochemistry, and expression pattern of A. annua Dbr1 point to its involvement in planta in the detoxification of 2-alkenals, which may be generated under oxidative stress conditions.

Functional Identification of Arabidopsis Stress Regulatory Genes Using the Controlled cDNA Overexpression System

Responses to environmental stresses in higher plants are controlled by a complex web of abscisic acid (ABA)-dependent and independent signaling pathways. To perform genetic screens for identification of novel Arabidopsis (Arabidopsis thaliana) loci involved in the control of abiotic stress responses, a complementary DNA (cDNA) expression library was created in a Gateway version of estradiol-inducible XVE binary vector (controlled cDNA overexpression system [COS]). The COS system was tested in three genetic screens by selecting for ABA insensitivity, salt tolerance, and activation of a stress-responsive ADH1-LUC (alcohol dehydrogenase-luciferase) reporter gene. Twenty-seven cDNAs conferring dominant, estradiol-dependent stress tolerance phenotype, were identified by polymerase chain reaction amplification and sequence analysis. Several cDNAs were recloned into the XVE vector and transformed recurrently into Arabidopsis, to confirm that the observed conditional phenotypes were due to their estradiol-dependent expression. Characterization of a cDNA conferring insensitivity to ABA in germination assays has identified the coding region of heat shock protein HSP17.6A suggesting its implication in ABA signal transduction. Screening for enhanced salt tolerance in germination and seedling growth assays revealed that estradiol-controlled overexpression of a 2-alkenal reductase cDNA confers considerable level of salt insensitivity. Screening for transcriptional activation of stress- and ABA-inducible ADH1-LUC reporter gene has identified the ERF/AP2-type transcription factor RAP2.12, which sustained high-level ADH1-LUC bioluminescence, enhanced ADH1 transcription rate, and increased ADH enzyme activity in the presence of estradiol. These data illustrate that application of the COS cDNA expression library provides an efficient strategy for genetic identification and characterization of novel regulators of abiotic stress responses.

Leukotriene B4 12-hydroxydehydrogenase/15-ketoprostaglandinΔ13-reductase (LTB4 12-HD/PGR) responsible for the reduction of a double-bond of the α,β-unsaturated ketone of an aryl propionic acid non-steroidal anti-inflammatory agent CS-670

CS-670 is a non-steroidal anti-inflammatory agent with an α,β-unsaturated ketone structure. It exerts its pharmacological activity after being transformed to the active metabolite (2S,1′R,2′S)-trans-alcohol. Two consecutive reductions are needed for the formation of the active metabolite, reduction of the double-bond of the α,β-unsaturated ketone moiety, followed by reduction of the resulting saturated ketone. The objective of the current study was to identify the enzyme responsible for reduction of the double-bond. An enzyme purified from rat liver cytosol as a single band on sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was analysed by a Mascot database search of nano-LC tandem mass spectrometry (MS/MS) data and the enzyme was identified as 2-alkenal reductase (EC 1.3.1.74), which is known as an β-nicotinamide adenine dinucleotide phosphate (NADPH)-dependent alkenal/one oxidoreductase and has a role for leukotriene B4 12-hydroxydehydrogenase/15-ketoprostaglandinΔ13-reductase (LTB4 12-HD/PGR). The identification was confirmed by cloning LTB4 12-HD/PGR cDNA from rat liver, expressing it in Escherichia coli, and characterizing the properties of the enzyme. The identity was further supported by the subcellular localization in cytosol, a cofactor requirement for NADPH, substrate specificity, and substantial inhibition by 15-ketoPGF2α, benzylideneacetophenone, indomethacin, and quercitrin. In addition to catalysing the biological reduction of eicosanoids, including prostaglandins, leukotrienes, and lipoxins, LTB4 12-HD/PGR was also determined to function as a xenobiotic-metabolizing enzyme.

Protection against Photooxidative Injury of Tobacco Leaves by 2-Alkenal Reductase. Detoxication of Lipid Peroxide-Derived Reactive Carbonyls

Degradation of lipid peroxides leads to the formation of cytotoxic 2-alkenals and oxenes (collectively designated reactive carbonyls). The novel NADPH-dependent oxidoreductase 2-alkenal reductase (AER; EC 1.3.1.74) from Arabidopsis (Arabidopsis thaliana), which is encoded by the gene At5g16970, catalyzes the reduction of the alpha,beta-unsaturated bond of reactive carbonyls, and hence is presumed to function in antioxidative defense in plants. Here we show that Arabidopsis AER (At-AER) has a broad substrate spectrum to biologically relevant reactive carbonyls. Besides 2-alkenals, the enzyme recognized as substrates the lipid peroxide-derived oxenes 9-oxo-octadeca-(10E),(12Z)-dienoic acid and 13-oxo-octadeca-(9E),(11Z)-dienoic acid, as well as the potent genotoxin 4-oxo-(2E)-nonenal, altogether suggesting AER has a key role in the detoxification of reactive carbonyls. To validate this conclusion by in vivo studies, transgenic tobacco (Nicotiana tabacum) plants that had 100- to 250-fold higher AER activity levels than control plants were generated. The engineered plants exhibited significantly less damage from either (1) the exogenously administered 4-hydroxy-(2E)-nonenal, (2) treatment with methyl viologen plus light, or (3) intense light. We further show that the At-AER protein fused with the Aequorea victoria green fluorescent protein localizes in cytosol and the nucleus in Bright-Yellow 2 cells. These results indicate that reactive carbonyls mediate photooxidative injury in leaf cells, and At-AER in the cytosol protects the cells by reducing the alpha,beta-unsaturated bond of the photoproduced reactive carbonyls.