Abdulsamie Hanano

Involvement of the caleosin/peroxygenase RD20 in the control of cell death during Arabidopsis responses to pathogens

Caleosins, mostly found in lipid droplets of seeds and leaves, are believed to play physiological roles through their enzymatic capacities to produce oxylipins. We recently identified the caleosin RD20 as a peroxygenase reducing endogenous fatty acid hydroperoxides into their corresponding alcohols. Such oxylipins confer tolerance to oxidative stress by decreasing reactive oxygen species accumulation and by minimizing cell death. RD20 expression being induced by pathogens, we have examined the mode of action of this caleosin in response to biotic stress. Plants overexpressing RD20 exhibited an alteration of their leaf cuticle wax components and an increased resistance to the fungus Alternaria brassicicola. Conversely, silencing RD20 led to an enhanced propagation of the fungus and to reduced severity of the damages caused by the inoculation of the bacteria Pseudomonas syringae pv tomato. We discuss these findings and propose that the major function of RD20 is to generate oxylipins modulating oxidative status and cell death.

A Caleosin-Like Protein with Peroxygenase Activity Mediates Aspergillus flavus Development, Aflatoxin Accumulation, and Seed Infection

ABSTRACT Caleosins are a small family of calcium-binding proteins endowed with peroxygenase activity in plants. Caleosin-like genes are present in fungi; however, their functions have not been reported yet. In this work, we identify a plant caleosin-like protein in Aspergillus flavus that is highly expressed during the early stages of spore germination. A recombinant purified 32-kDa caleosin-like protein supported peroxygenase activities, including co-oxidation reactions and reduction of polyunsaturated fatty acid hydroperoxides. Deletion of the caleosin gene prevented fungal development. Alternatively, silencing of the gene led to the increased accumulation of endogenous polyunsaturated fatty acid hydroperoxides and antioxidant activities but to a reduction of fungal growth and conidium formation. Two key genes of the aflatoxin biosynthesis pathway, aflR and aflD, were downregulated in the strains in which A. flavus PXG (AfPXG) was silenced, leading to reduced aflatoxin B1 production in vitro. Application of caleosin/peroxygenase-derived oxylipins restored the wild-type phenotype in the strains in which AfPXG was silenced. PXG-deficient A. flavus strains were severely compromised in their capacity to infect maize seeds and to produce aflatoxin. Our results uncover a new branch of the fungal oxylipin pathway and may lead to the development of novel targets for controlling fungal disease.

Biochemical, Transcriptional, and Bioinformatic Analysis of Lipid Droplets from Seeds of Date Palm (Phoenix dactylifera L.) and Their Use as Potent Sequestration Agents against the Toxic Pollutant, 2,3,7,8-Tetrachlorinated Dibenzo-p-Dioxin

Contamination of aquatic environments with dioxins, the most toxic group of persistent organic pollutants (POPs), is a major ecological issue. Dioxins are highly lipophilic and bioaccumulate in fatty tissues of marine organisms used for seafood where they constitute a potential risk for human health. Lipid droplets (LDs) purified from date palm, Phoenix dactylifera, seeds were characterized and their capacity to extract dioxins from aquatic systems was assessed. The bioaffinity of date palm LDs toward 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most toxic congener of dioxins was determined. Fractioned LDs were spheroidal with mean diameters of 2.5 µm, enclosing an oil-rich core of 392.5 mg mL-1. Isolated LDs did not aggregate and/or coalesce unless placed in acidic media and were strongly associated with three major groups of polypeptides of relative mass 32–37, 20–24, and 16–18 kDa. These masses correspond to the LD-associated proteins, oleosins, caleosins, and steroleosins, respectively. Efficient partitioning of TCDD into LDs occurred with a coefficient of log KLB/w,TCDD = 7.528 ± 0.024; it was optimal at neutral pH and was dependent on the presence of the oil-rich core, but was independent of the presence of LD-associated proteins. Bioinformatic analysis of the date palm genome revealed nine oleosin-like, five caleosin-like, and five steroleosin-like sequences, with predicted structures having putative lipid-binding domains that match their LD stabilizing roles and use as bio-based encapsulation systems. Transcriptomic analysis of date palm seedlings exposed to TCDD showed strong up-regulation of several caleosin and steroleosin genes, consistent with increased LD formation. The results suggest that the plant LDs could be used in ecological remediation strategies to remove POPs from aquatic environments. Recent reports suggest that several fungal and algal species also use LDs to sequester both external and internally derived hydrophobic toxins, which indicates that our approach could be used as a broader biomimetic strategy for toxin removal.

Saccharomyces cerevisiae SHSY detoxifies petroleum n-alkanes by an induced CYP52A58 and an enhanced order in cell surface hydrophobicity.

Environmental hydrocarbon contamination has a serious hazard to human health. Alkanes, the major component of hydrocarbons, can be consumed by various species of yeast. We previously identified a new strain SHSY of Saccharomyces cerevisiae with a remarkable ability to utilize the petroleum crude-oil (PCO) in aqueous solution. The current study demonstrated that the n-alkanes-assimilation activity of S. cerevisiae SHSY was related to an induced microsomal protein of 59 kDa approximately. The identified ORF encoded a protein of 517 amino acids and shared 93% sequence identity with an alkane-inducible hydroxylase CYP52A53 isolated from Scheffersomyces stipitis CBS. It was therefore referred as CYP52A58. The catalytic activity of the recombinant CYP52A58 was confirmed by the hydroxylation of n-alkanes, it showed an optimal mono-terminal hydroxylation activity toward n-hexadecane. Moreover, the ability of the yeast to use n-alkanes was accompanied with an increasing level in cell wall mannoproteins. Two differential protein bands were detected in the mannoproteins extracted from PCO-grown yeast. In parallel, a significant increase in the fatty acids content with a high degree of unsaturation was subsequently detected in the PCO-grown yeast. This study characterizes a safe and potential microorganism to remove n-alkanes from the aquatic environment.

Differential tissue accumulation of 2,3,7,8-Tetrachlorinated dibenzo-p-dioxin in Arabidopsis thaliana affects plant chronology, lipid metabolism and seed yield.

BackgroundDioxins are one of the most toxic groups of persistent organic pollutants. Their biotransmission through the food chain constitutes a potential risk for human health. Plants as principal actors in the food chain can play a determinant role in removing dioxins from the environment. Due to the lack of data on dioxin/plant research, this study sets out to determine few responsive reactions adopted by Arabidopsis plant towards 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most toxic congener of dioxins.ResultsUsing a high resolution gas chromatography/mass spectrometry, we demonstrated that Arabidopsis plant uptakes TCDD by the roots and accumulates it in the vegetative parts in a tissue-specific manner. TCDD mainly accumulated in rosette leaves and mature seeds and less in stem, flowers and immature siliques. Moreover, we observed that plants exposed to high doses of TCDD exhibited a delay in flowering and yielded fewer seeds of a reduced oil content with a low vitality. A particular focus on the plant fatty acid metabolism showed that TCDD caused a significant reduction in C18-unsaturated fatty acid level in plant tissues. Simultaneously, TCDD induced the expression of 9-LOX and 13-LOX genes and the formation of their corresponding hydroperoxides, 9- and 13-HPOD as well as 9- or 13-HPOT, derived from linoleic and linolenic acids, respectively.ConclusionsThe current work highlights a side of toxicological effects resulting in the administration of 2,3,7,8-TCDD on the Arabidopsis plant. Similarly to animals, it seems that plants may accumulate TCDD in their lipids by involving few of the FA-metabolizing enzymes for sculpting a specific oxylipins “signature” typified to plant TCDD-tolerance. Together, our results uncover novel responses of Arabidopsis to dioxin, possibly emerging to overcome its toxicity.

Removal of petroleum-crude oil from aqueous solution by Saccharomyces cerevisiae SHSY strain necessitates at least an inducible CYP450ALK homolog gene.

Petroleum crude‐oil (PCO) components are known to be mutagenic or carcinogenic, and their contamination in soil and aquifer is of great environmental concern. PCO could be degraded by bacteria, fungi, and yeast. In yeast, the family CYP52 (P450ALKs) of Cytochrome P450 was described as n‐alkane‐degrading enzymes. In this study, we isolated a new strain SHSY of Saccharomyces able to grow on hydrocarbons compounds. Morphological and molecular characterization led to identify the isolated yeast SHSY as a Saccharomyces cerevisiae. SHSY strain had a remarkable ability to tolerate a high concentration of PCO and use it as a carbon source. A significant relationship was established between the increase in biomass (42.46 ± 1.01‐fold) and the disappearance of the crude oil (72.34%) in an aqueous solution. A 690‐bp amplicon corresponding to a high conserved region of known CYP450ALK genes was amplified in the genomic DNA of SHSY strain. The sequence of the amplified fragment shared a high identity (71.8%) with CYP52A3 gene of Pichia stipites. The expression of CYP52A3 homolog gene was induced and the expression of both InoP2/InoP4 transcription factor genes in SHSY was stimulated in the presence of PCO. The identified strain SHSY of S. cerevisiae presents an interesting model to minimize the mixed toxicity of PCO in polluted environmental sites.

Biochemical, Molecular, and Transcriptional Highlights of the Biosynthesis of an Effective Biosurfactant Produced by Bacillus safensis PHA3, a Petroleum-Dwelling Bacteria

Petroleum crude oil (PCO)-dwelling microorganisms have exceptional biological capabilities to tolerate the toxicity of petroleum contaminants and are therefore promising emulsifier and/or degraders of PCO. This study describes a set of PCO-inhabiting bacterial species, one of which, identified as Bacillus safensis PHA3, produces an efficient biosurfactant which was characterized as a glycolipid. Fourier transform infrared spectrometer, nuclear magnetic resonance, Thin layer chromatography, HPLC, and GC-MS analysis of the purified biosurfactant revealed that the extracted molecule under investigation is likely a mannolipid molecule with a hydrophilic part as mannose and a hydrophobic part as hexadecanoic acid (C16:0). The data reveal that: (i) PHA3 is a potential producer of biosurfactant (9.8 ± 0.5 mg mL-1); (ii) pre-adding 0.15% of the purified glycolipid enhanced the degradation of PCO by approximately 2.5-fold; (iii) the highest emulsifying activity of biosurfactant was found against the PCO and the lowest was against the naphthalene; (iv) the optimal PCO-emulsifying activity was found at 30–60°C, pH 8 and a high salinity. An orthologous gene encodes a putative β-diglucosyldiacylglycerol synthase (β-DGS) was identified in PHA3 and its transcripts were significantly up-regulated by exogenous PAHs, i.e., pyrene and benzo(e)pyrene but much less by mid-chain n-alkanes (ALKs) and fatty acids. Subsequently, the accumulation of β-DGS transcripts coincided with an optimal growth of bacteria and a maximal accumulation of the biosurfactant. Of particular interest, we found that PHA3 actively catalyzed the degradation of PAHs notably the pyrene and benzo(e)pyrene but was much less effective in the mono-terminal oxidation of ALKs. Such characteristics make Bacillus safensis PHA3 a promising model for enhanced microbial oil recovery and environmental remediation.

Specific Caleosin/Peroxygenase and Lipoxygenase Activities Are Tissue-Differentially Expressed in Date Palm (Phoenix dactylifera L.) Seedlings and Are Further Induced Following Exposure to the Toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin

Two caleosin/peroxygenase isoforms from date palm, Phoenix dactylifera L., PdCLO2 and PdCLO4, were characterized with respect to their tissue expression, subcellular localization, and oxylipin pathway substrate specificities in developing seedlings. Both PdCLO2 and PdCLO4 had peroxygenase activities that peaked at the mid-stage (radicle length of 2.5 cm) of seedling growth and were associated with the lipid droplet (LD) and microsomal fractions. Recombinant PdCLO2 and PdCLO4 proteins heterologously expressed in yeast cells were localized in both LD and microsomal fractions. Each of the purified recombinant proteins exhibited peroxygenase activity but they were catalytically distinct with respect to their specificity and product formation from fatty acid epoxide and hydroxide substrates. We recently showed that date palm CLO genes were upregulated following exposure to the potent toxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Hanano et al., 2016), and we show here that transcripts of 9- and 13-lipoxygenase (LOX) genes were also induced by TCDD exposure. At the enzyme level, 9-LOX and 13-LOX activities were present in a range of seedling tissues and responded differently to TCDD exposure, as did the 9- and 13-fatty acid hydroperoxide reductase activities. This demonstrates that at least two branches of the oxylipin pathway are involved in responses to the environmental organic toxin, TCDD in date palm.

The Peroxygenase Activity of the Aspergillus flavus Caleosin, AfPXG, Modulates the Biosynthesis of Aflatoxins and Their Trafficking and Extracellular Secretion via Lipid Droplets

Aflatoxins (AF) are highly detrimental to human and animal health. We recently demonstrated that the Aspergillus flavus caleosin, AfPXG, had peroxygenase activity and mediated fungal development and AF accumulation. We now report the characterization of an AfPXG-deficient line using reference strain NRRL3357. The resulting fungal phenotype included a severe decrease in mycelium growth, failure to sporulate, and reduced AF production. Increasing cellular oxidative status by administration of hydrogen peroxide and cumene hydroperoxide did not restore the AfPXG-deficient phenotype, which suggests that AfPXG-deficiency is not directly related to oxidative stress. To investigate possible alternative roles of AfPXG, a gain of function approach was used to overexpress AfPXG, with the reporter gene Gfp, in an AfPXG-deficient line, termed AfPXG+. The resulting phenotype included elevated numbers of stable lipid droplets (LDs) plus enhanced AF production. Highly purified LDs from AfPXG+ cultures sequestered AF and this ability was positively correlated with overall LD number. Site-specific mutagenesis of AfPXG to delete Histidine 85 (AfPXGHis85), a residue essential for its catalytic activity, or deletion of the putative LD targeting domain (AfPXGD126-140), showed that AfPXG-peroxygenase activity was required for AF biosynthesis and that integration of AF into LDs was required for their export via a LD-dependent pathway. Ectopic expression in fungal cells of the plant LD-associated protein, oleosin, also resulted in both additional LD accumulation and enhanced AF secretion. These results suggest that both fungal LDs and their associated caleosin proteins are intimately involved in the biosynthesis, trafficking, and secretion of AF.

Identification of a dioxin-responsive oxylipin signature in roots of date palm: involvement of a 9-hydroperoxide fatty acid reductase, caleosin/peroxygenase PdPXG2

Dioxins are highly hazardous pollutants that have well characterized impacts on both animal and human health. However, the biological effects of dioxins on plants have yet to be described in detail. Here we describe a dioxin-inducible caleosin/peroxygenase isoform, PdPXG2, that is mainly expressed in the apical zone of date palm roots and specifically reduces 9-hydroperoxide fatty acids. A characteristic spectrum of 18 dioxin-responsive oxylipin (DROXYL) congeners was also detected in date palm roots after exposure to dioxin. Of particular interest, six oxylipins, mostly hydroxy fatty acids, were exclusively formed in response to TCDD. The DROXYL signature was evaluated in planta and validated in vitro using a specific inhibitor of PdPXG2 in a root-protoplast system. Comparative analysis of root suberin showed that levels of certain monomers, especially the mono-epoxides and tri-hydroxides of C16:3 and C18:3, were significantly increased after exposure to TCDD. Specific inhibition of PdPXG2 activity revealed a positive linear relationship between deposition of suberin in roots and their permeability to TCDD. The results highlight the involvement of this peroxygenase in the plant response to dioxin and suggest the use of dioxin-responsive oxylipin signatures as biomarkers for plant exposure to this important class of xenobiotic contaminants.