Alkenal Reductase Research Articles

Developing a Nicotiana benthamiana transgenic platform for high-value diterpene production and candidate gene evaluation.

SummaryTo engineer Nicotiana benthamiana to produce novel diterpenoids, we first aimed to increase production of the diterpenoid precursor geranylgeranyl pyrophosphate (GGPP) by up‐regulation of key genes of the non‐mevalonate (MEP) pathway sourced from Arabidopsis thaliana. We used transient expression to evaluate combinations of the eight MEP pathway genes plus GGPP synthase and a Jatropha curcas casbene synthase (JcCAS) to identify an optimal combination for production of casbene from GGPP. AtDXS and AtHDR together with AtGGPPS and JcCAS gave a 410% increase in casbene production compared to transient expression of JcCAS alone. This combination was cloned into a single construct using the MoClo toolkit, and stably integrated into the N. benthamiana genome. We also created multigene constructs for stable transformation of two J. curcas cytochrome P450 genes, JcCYP726A20 and JcCYP71D495 that produce the more complex diterpenoid jolkinol C from casbene when expressed transiently with JcCAS in N. benthamiana. Stable transformation of JcCYP726A20, JcCYP71D495 and JcCAS did not produce any detectable jolkinol C until these genes were co‐transformed with the optimal set of precursor‐pathway genes. One such stable homozygous line was used to evaluate by transient expression the involvement of an ‘alkenal reductase’‐like family of four genes in the further conversion of jolkinol C, leading to the demonstration that one of these performs reduction of the 12,13‐double bond in jolkinol C. This work highlights the need to optimize precursor supply for production of complex diterpenoids in stable transformants and the value of such lines for novel gene discovery.

Luminescence imaging of leaf damage induced by lipid peroxidation products and its modulation by β‐cyclocitral

Lipid peroxidation is a primary event associated with oxidative stress in plants. This phenomenon secondarily generates bioactive and/or toxic compounds such as reactive carbonyl species (RCS), phytoprostanes and phytofurans, as confirmed here in Arabidopsis plants exposed to photo-oxidative stress conditions. We analyzed the effects of exogenous applications of secondary lipid oxidation products on Arabidopsis plants by luminescence techniques. Oxidative damage to attached leaves was measured by autoluminescence imaging, using a highly-sensitive CCD camera, and the activity of the detoxification pathway, dependent on the transcription regulator SCARECROW-LIKE 14 (SCL14), was monitored with a bioluminescent line expressing the firefly LUCIFERASE (LUC) gene under the control of the ALKENAL REDUCTASE (AER) gene promoter. We identified 4-hydroxynonenal (HNE), and to a lesser extent 4-hydroxyhexenal (HHE), as highly reactive compounds that are harmful to leaves and can trigger AER gene expression, contrary to other RCS (pentenal, hexenal) and to isoprostanoids. Although the levels of HNE and other RCS were enhanced in the SCL14-deficient mutant (scl14), exogenously applied HNE was similarly damaging to this mutant, its wild-type parent and a SCL14-overexpressing transgenic line (OE:SCL14). However, strongly boosting the SCL14 detoxification pathway and AER expression by a pre-treatment of OE:SCL14 with the signaling apocarotenoid β-cyclocitral cancelled the damaging effects of HNE. Conversely, in the scl14 mutant, the effects of β-cyclocitral and HNE were additive, leading to enhanced leaf damage. These results indicate that the cellular detoxification pathway induced by the low-toxicity β-cyclocitral targets highly toxic compounds produced during lipid peroxidation, reminiscent of a safener-type mode of action. This article is protected by copyright. All rights reserved.

Endogenous Biosynthesis of S-Nitrosoglutathione From Nitro-Fatty Acids in Plants.

Nitro-fatty acids (NO2-FAs) are novel molecules resulting from the interaction of unsaturated fatty acids and nitric oxide (NO) or NO-related molecules. In plants, it has recently been described that NO2-FAs trigger an antioxidant and a defence response against stressful situations. Among the properties of NO2-FAs highlight the ability to release NO therefore modulating specific protein targets through post-translational modifications (NO-PTMs). Thus, based on the capacity of NO2-FAs to act as physiological NO donors and using high-accuracy mass-spectrometric approaches, herein, we show that endogenous nitro-linolenic acid (NO2-Ln) can modulate S-nitrosoglutathione (GSNO) biosynthesis in Arabidopsis. The incubation of NO2-Ln with GSH was analyzed by LC-MS/MS and the in vitro synthesis of GSNO was noted. The in vivo confirmation of this behavior was carried out by incubating Arabidopsis plants with 15N-labeled NO2-Ln throughout the roots, and 15N-labeled GSNO (GS15NO) was detected in the leaves. With the aim to go in depth in the relation of NO2-FA and GSNO in plants, Arabidopsis alkenal reductase mutants (aer mutants) which modulate NO2-FAs levels were used. Our results constitute the first evidence of the modulation of a key NO biological reservoir in plants (GSNO) by these novel NO2-FAs, increasing knowledge about S-nitrosothiols and GSNO-signaling pathways in plants.

Amine oxidase 3 is a novel pro-inflammatory marker of oxidative stress in peritoneal endometriosis lesions

Endometriosis is a common gynaecological disease of women in reproductive age, and is thought to arise from retrograde menstruation and implantation of endometrial tissue, mostly into the peritoneal cavity. The condition is characterized by a chronic, unresolved inflammatory process thereby contributing to pain as cardinal symptom in endometriosis. Elevated reactive oxygen species (ROS) and oxidative stress have been postulated as factors in endometriosis pathogenesis. We here set out for a systematic study to identify novel mechanisms and pathways relating to oxidative stress in ectopic peritoneal lesions. Using combined proteomic and transcriptomic approaches, we identified novel targets including upregulated pro-oxidative enzymes, such as amine oxidase 3/vascular adhesion protein 1 (AOC3/VAP1) as well as downregulated protective factors, in particular alkenal reductase PTGR1 and methionine sulfoxide reductase. Consistent with an altered ROS landscape, we observed hemoglobin / iron overload, ROS production and lipid peroxidation in ectopic lesions. ROS-derived 4-hydroxy-2-nonenal induced interleukin IL-8 release from monocytes. Notably, AOC3 inhibitors provoked analgesic effects in inflammatory pain models in vivo, suggesting potential translational applicability.

Reactive Carbonyl Species Function as Signal Mediators Downstream of H2O2 Production and Regulate [Ca2+]cyt Elevation in ABA Signal Pathway in Arabidopsis Guard Cells.

We have demonstrated that reactive carbonyl species (RCS) function as an intermediate downstream of hydrogen peroxide (H2O2) production in abscisic acid (ABA) signaling for stomatal closure in guard cells using transgenic tobacco plants overexpressing alkenal reductase. We investigated the conversion of the RCS production into downstream signaling events in the guard cells. Both ABA and H2O2 induced production of the RCS, such as acrolein and 4-hydroxy-(E)-2-nonenal (HNE), in epidermal tissues of wild-type Arabidopsis thaliana plants. Application of the RCS scavengers, carnosine and pyridoxamine, did not affect the ABA-induced H2O2 production but inhibited the ABA- and H2O2-induced stomatal closure. Both acrolein and HNE induced stomatal closure in a plasma membrane NAD(P)H oxidase mutant atrbohD atrbohF as well as in the wild type, but not in a calcium-dependent kinase mutant cpk6. Acrolein activated plasma membrane Ca2+-permeable cation channels, triggered cytosolic free Ca2+ concentration ([Ca2+]cyt) elevation, and induced stomatal closure accompanied by depletion of glutathione in the guard cells. These results suggest that RCS production is a signaling event between the ROS production and [Ca2+]cyt elevation during guard cell ABA signaling.

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.

Crystal Structure of the Chloroplastic Oxoene Reductase ceQORH from Arabidopsis thaliana.

Enzymatic and non-enzymatic peroxidation of polyunsaturated fatty acids give rise to accumulation of aldehydes, ketones, and α,β-unsaturated carbonyls of various lengths, known as oxylipins. Oxylipins with α,β-unsaturated carbonyls are reactive electrophile species and are toxic. Cells have evolved several mechanisms to scavenge reactive electrophile oxylipins and decrease their reactivity such as by coupling with glutathione, or by reduction using NAD(P)H-dependent reductases and dehydrogenases of various substrate specificities. Plant cell chloroplasts produce reactive electrophile oxylipins named γ-ketols downstream of enzymatic lipid peroxidation. The chloroplast envelope quinone oxidoreductase homolog (ceQORH) from Arabidopsis thaliana was previously shown to reduce the reactive double bond of γ-ketols. In marked difference with its cytosolic homolog alkenal reductase (AtAER) that displays a high activity toward the ketodiene 13-oxo-9(Z),11(E)-octadecadienoic acid (13-KODE) and the ketotriene 13-oxo-9(Z), 11(E), 15(Z)-octadecatrienoic acid (13-KOTE), ceQORH binds, but does not reduce, 13-KODE and 13-KOTE. Crystal structures of apo-ceQORH and ceQORH bound to 13-KOTE or to NADP+ and 13-KOTE have been solved showing a large ligand binding site, also observed in the structure of the cytosolic alkenal/one reductase. Positioning of the α,β-unsaturated carbonyl of 13-KOTE in ceQORH-NADP+-13-KOTE, far away from the NADP+ nicotinamide ring, provides a rational for the absence of activity with the ketodienes and ketotrienes. ceQORH is a monomeric enzyme in solution whereas other enzymes from the quinone oxidoreductase family are stable dimers and a structural explanation of this difference is proposed. A possible in vivo role of ketodienes and ketotrienes binding to ceQORH is also discussed.

Transcriptomic profiling of linolenic acid-responsive genes in ROS signaling from RNA-seq data in Arabidopsis

Linolenic acid (Ln) released from chloroplast membrane galactolipids is a precursor of the phytohormone jasmonic acid (JA). The involvement of this hormone in different plant biological processes, such as responses to biotic stress conditions, has been extensively studied. However, the role of Ln in the regulation of gene expression during abiotic stress situations mediated by cellular redox changes and/or by oxidative stress processes remains poorly understood. An RNA-seq approach has increased our knowledge of the interplay among Ln, oxidative stress and ROS signaling that mediates abiotic stress conditions. Transcriptome analysis with the aid of RNA-seq in the absence of oxidative stress revealed that the incubation of Arabidopsis thaliana cell suspension cultures (ACSC) with Ln resulted in the modulation of 7525 genes, of which 3034 genes had a 2-fold-change, being 533 up- and 2501 down-regulated genes, respectively. Thus, RNA-seq data analysis showed that an important set of these genes were associated with the jasmonic acid biosynthetic pathway including lypoxygenases (LOXs) and Allene oxide cyclases (AOCs). In addition, several transcription factor families involved in the response to biotic stress conditions (pathogen attacks or herbivore feeding), such as WRKY, JAZ, MYC, and LRR were also modified in response to Ln. However, this study also shows that Ln has the capacity to modulate the expression of genes involved in the response to abiotic stress conditions, particularly those mediated by ROS signaling. In this regard, we were able to identify new targets such as galactinol synthase 1 (GOLS1), methionine sulfoxide reductase (MSR) and alkenal reductase in ACSC. It is therefore possible to suggest that, in the absence of any oxidative stress, Ln is capable of modulating new sets of genes involved in the signaling mechanism mediated by additional abiotic stresses (salinity, UV and high light intensity) and especially in stresses mediated by ROS.

CmRT1, a Ty3-gypsy-like retrotransposon in Castanea mollissima,is associated with a short-catkin mutation

SummaryRetrotransposons are major components of the genomes of most eukaryotic organisms and have resulted in the introduction of desirable traits in many crops, including fruit trees. Here, we describe a Ty3-gypsy-like retrotransposon associated with a short-catkin mutant in Chinese chestnut (Castanea mollissima), resulting in catkins that are < 20% the length of normal staminate catkins. A partial sequence of the retrotransposon, named CmRT1, detected by amplified fragment length polymorphism (AFLP) analysis, and its complete sequence were determined from the genome of Chinese chestnut (Castanea mollissima) using improved Tail-PCR. CmRT1 was 10,067 bp in length and shared high homology in its predicted amino acid sequence and motifs with other Ty3/gypsy-like retrotransposons. The 5’ long terminal repeat (LTR) of CmRT1 contained a TATA box and several cis-elements that were predicted to be important for processes involving abscisic acid, gibberellic acid, and auxins and in stress-mediated responses. Further characterisation of the transposition event that led to the short-catkin phenotype was performed using two pairs of primers that aligned with the flanking region of the LTRs. The expected PCR bands were observed only in genomic DNA from plants that showed the mutation. Finally, cloning and real-time qPCR analysis of an NADP-dependent alkenal double-bond reductase (CmADBR) target gene that was adjacent to CmRT1, revealed that CmADBR expression was significantly down-regulated in the short-catkin mutant. Taken together, these results suggest that the CmRT1 retrotransposon is responsible for the short-catkin phenotype.

NADPH-dependent Reductases Involved in the Detoxification of Reactive Carbonyls in Plants

Reactive carbonyls, especially α,β-unsaturated carbonyls produced through lipid peroxidation, damage biomolecules such as proteins and nucleotides; elimination of these carbonyls is therefore essential for maintaining cellular homeostasis. In this study, we focused on an NADPH-dependent detoxification of reactive carbonyls in plants and explored the enzyme system involved in this detoxification process. Using acrolein (CH(2) = CHCHO) as a model α,β-unsaturated carbonyl, we purified a predominant NADPH-dependent acrolein-reducing enzyme from cucumber leaves, and we identified the enzyme as an alkenal/one oxidoreductase (AOR) catalyzing reduction of an α,β-unsaturated bond. Cloning of cDNA encoding AORs revealed that cucumber contains two distinct AORs, chloroplastic AOR and cytosolic AOR. Homologs of cucumber AORs were found among various plant species, including Arabidopsis, and we confirmed that a homolog of Arabidopsis (At1g23740) also had AOR activity. Phylogenetic analysis showed that these AORs belong to a novel class of AORs. They preferentially reduced α,β-unsaturated ketones rather than α,β-unsaturated aldehydes. Furthermore, we selected candidates of other classes of enzymes involved in NADPH-dependent reduction of carbonyls based on the bioinformatic information, and we found that an aldo-keto reductase (At2g37770) and aldehyde reductases (At1g54870 and At3g04000) were implicated in the reduction of an aldehyde group of saturated aldehydes and methylglyoxal as well as α,β-unsaturated aldehydes in chloroplasts. These results suggest that different classes of NADPH-dependent reductases cooperatively contribute to the detoxification of reactive carbonyls.