Conversion of methylglyoxal to acetol by Escherichia coli aldo-keto reductases

Methylglyoxal (MG) is a toxic metabolite known to accumulate in various cell types. We detected in vivo conversion of MG to acetol in MG-accumulating Escherichia coli cells by use of (1)H nuclear magnetic resonance ((1)H-NMR) spectroscopy. A search for homologs of the mammalian aldo-keto reductases (AKRs), which are known to exhibit activity to MG, revealed nine open reading frames from the E. coli genome. Based on both sequence similarities and preliminary characterization with (1)H-NMR for crude extracts of the corresponding mutant strains, we chose five genes, yafB, yqhE, yeaE, yghZ, and yajO, for further study. Quantitative assessment of the metabolites produced in vitro from the crude extracts of these mutants and biochemical study with purified AKRs indicated that the yafB, yqhE, yeaE, and yghZ genes are involved in the conversion of MG to acetol in the presence of NADPH. When we assessed their in vivo catalytic activities by creating double mutants, all of these genes except for yqhE exhibited further sensitivities to MG in a glyoxalase-deficient strain. The results imply that the glutathione-independent detoxification of MG can occur through multiple pathways, consisting of yafB, yqhE, yeaE, and yghZ genes, leading to the generation of acetol.     

Correlations between Photodegradation of Bisretinoid Constituents of Retina and Dicarbonyl Adduct Deposition

Non-enzymatic collagen cross-linking and carbonyl adduct deposition are features of Bruch''s membrane aging in the eye, and disturbances in extracellular matrix turnover are considered to contribute to Bruch''s membrane thickening. Because bisretinoid constituents of the lipofuscin of retinal pigment epithelial (RPE) cells are known to photodegrade to mixtures of aldehyde-bearing fragments and small dicarbonyls (glyoxal (GO) and methylglyoxal (MG)), we investigated RPE lipofuscin as a source of the reactive species that covalently modify protein side chains. Abca4^−/− and Rdh8^−/−/Abca4^−/− mice that are models of accelerated bisretinoid formation were studied and pre-exposure of mice to 430 nm light enriched for dicarbonyl release by bisretinoid photodegradation. MG protein adducts were elevated in posterior eyecups of mutant mice, whereas carbonylation of an RPE-specific protein was observed in Abca4^−/− but not in wild-type mice under the same conditions. Immunolabeling of cryostat-sectioned eyes harvested from Abca4^−/− mice revealed that carbonyl adduct deposition in Bruch''s membrane was accentuated. Cell-based assays corroborated these findings in mice. Moreover, the receptor for advanced glycation end products that recognizes MG and GO adducts and glyoxylase 1 that metabolizes MG and GO were up-regulated in Abca4^−/− mice. Additionally, in acellular assays, peptides were cross-linked in the presence of A2E (adduct of two vitamin A aldehyde and ethanolamine) photodegradation products, and in a zymography assay, reaction of collagen IV with products of A2E photodegradation resulted in reduced cleavage by the matrix metalloproteinases MMP2 and MMP9. In conclusion, these mechanistic studies demonstrate a link between the photodegradation of RPE bisretinoid fluorophores and aging changes in underlying Bruch''s membrane that can confer risk of age-related macular degeneration.     

Identifying a Polymorphic ‘Switch’ That Influences miRNAs' Regulation of a Myasthenia Gravis Risk Pathway

The significant roles of genetic variants in myasthenia gravis (MG) pathogenesis have been demonstrated in many studies, and recently it has been revealed that aberrant level/regulation of microRNAs (miRNAs) might contribute to the initiation and progression of MG. However, the dysfunction of miRNA associated with single nucleotide polymorphisms (miRSNPs) has not been well investigated in MG. In this study, we created a contemporary catalog of 89 MG risk genes via manual literature-mining. Based on this risk gene catalog, we obtained 18 MG risk pathways. Furthermore, we identified 93 miRNAs that target MG risk pathways and revealed the miRSNPs ‘switches’ in miRNA regulation in the MG risk pathways by integrating the database information of miRSNPs. We also constructed a miRNA-mediated SNP switching pathway network (MSSPN) to intuitively analyze miRNA regulation of MG risk pathways and the relationship of the polymorphism ‘switch’ with these changes in regulation. Moreover, we carried out in-depth dissection on the correlation between hsa05200 (pathway in cancer) and MG development, and elaborated the significance of 4 high-risk genes. By network analysis and literature mining, we proposed a potential mechanism of miRSNPs→gene→pathway effects on MG pathogenesis, especially for rs28457673 (miR-15/16/195/424/497 family)→IGF1R→hsa05200 (pathway in cancer). Therefore, our studies have revealed a functional role for genetic modulators in MG pathogenesis at a systemic level, which could be informative for further miRNA and miRSNPs studies in MG.     

AKT Signaling as a Novel Factor Associated with In Vitro Resistance of Human AML to Gemtuzumab Ozogamicin

Gemtuzumab ozogamicin (GO), an immunoconjugate between an anti-CD33 antibody and a calicheamicin-γ₁ derivative, induces remissions and improves survival in a subset of patients with acute myeloid leukemia (AML). As the mechanisms underlying GO and calicheamicin-γ₁ resistance are incompletely understood, we herein used flow cytometry-based single cell network profiling (SCNP) assays to study cellular responses of primary human AML cells to GO. Our data indicate that the extent of DNA damage is quantitatively impacted by CD33 expression and drug efflux activity. However, although DNA damage is required for GO-induced cytotoxicity, it is not sufficient for effective cell kill, suggesting that downstream anti-apoptotic pathways may function as relevant resistance mechanisms. Supporting this notion, we found activated PI3K/AKT signaling to be associated with GO resistance in vitro in primary AML cells. Consistently, the investigational AKT inhibitor MK-2206 significantly sensitized various human AML cells to GO or free calicheamicin-γ₁ with particularly pronounced effects in otherwise GO or free calicheamicin-γ₁ -resistant cells. Likewise, MK-2206 also sensitized primary AML cells to calicheamicin-γ₁. Together, our findings illustrate the capacity of SCNP assays to discover chemotherapy-related biological pathways and signaling networks relevant to GO-induced genotoxic stress. The identification of AKT signaling as being associated with GO resistance in vitro may provide a novel approach to improve the in vivo efficacy of GO/calicheamicin-γ₁ and, by extrapolation, other DNA damage-based therapeutics.     

2D [<Superscript>1</Superscript>H,<Superscript>13</Superscript>C] NMR study of carbon fluxes during glucose utilization by <Emphasis Type="Italic">Escherichia coli</Emphasis> MG1655

Carbon fluxes through main pathways of glucose utilization in Escherichia coli cells-glycolysis, pentose phosphate pathway (PPP), and Enther-Doudoroff pathway (EDP)—were studied. Their ratios were analyzed in E. coli strains MG1655, MG1655Δ(edd-eda), MG1655Δ(zwf, edd-eda), and MG1655Δ(pgi, edd-eda). It was shown that the carbon flux through glycolysis was the main route of glucose utilization, averaging ca. 80%. Inactivation of EDP did not affect growth parameters. Nevertheless, it altered carbon fluxes through the tricarboxylic acid cycles and energy metabolism in the cell. Inactivation of PPP decreased growth rate to a lesser degree than glycolysis inactivation.     

Crystal Structure and Comparative Functional Analyses of a Mycobacterium Aldo-Keto Reductase

Aldo-keto reductases (AKRs) are a large superfamily of NADPH-dependent enzymes that catalyze the reduction of aldehydes, aldoses, dicarbonyls, steroids, and monosaccharides. While their precise physiological role is generally unknown, AKRs are nevertheless involved in the detoxification of a broad range of toxic metabolites. Mycobacteria contain a number of AKRs, the majority of which are uncharacterised. Here, we report the 1.9 and 1.6 Å resolution structures of the apoenzyme and NADPH-bound forms, respectively, of an AKR (MSMEG_2407) from Mycobacterium smegmatis, a close homologue of the M. tuberculosis enzyme Rv2971, whose function is essential to this bacterium. MSMEG_2407 adopted the triosephosphate isomerase (α/β)₈-barrel fold exhibited by other AKRs. MSMEG_2407 (AKR5H1) bound NADPH via an induced-fit mechanism, in which the NADPH was ligated in an extended fashion. Polar-mediated interactions dominated the interactions with the cofactor, which is atypical of the mode of NADPH binding within the AKR family. Moreover, the nicotinamide ring of NADPH was disordered, and this was attributed to the lack of an “AKR-conserved” bulky residue within the nicotinamide-binding cavity of MSMEG_2407. Enzymatic characterisation of MSMEG_2407 and Rv2971 identified dicarbonyls as a preferred substrate family for hydrolysis, and the frontline antituberculosis drug isoniazid (INH) was shown to inhibit the enzyme activity of both recombinant MSMEG_2407 and Rv2971. However, differences between the affinities of MSMEG_2407 and Rv2971 for dicarbonyls and INH were observed, and this was attributable to amino acid substitutions within the cofactor- and substrate-binding sites. The structures of MSMEG_2407 and the accompanying biochemical characterisation of MSMEG_2407 and Rv2971 provide insight into the structure and function of AKRs from mycobacteria.     

Identification and functional characterization of four novel aldo/keto reductases in <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC 7120 by integrating wet lab with in silico approaches

Aldo/keto reductases (AKRs) constitute a multitasking protein family that catalyzes diverse metabolic transformations including detoxification of stress generated reactive aldehydes. Yet this important protein family is poorly understood particularly in cyanobacteria, the ecologically most diverse and significant group of micro-organisms. Present study is an attempt to characterize all putative AKRs of Anabaena sp. PCC 7120. In silico analysis, it revealed the presence of at least four putative AKRs in Anabaena PCC7120 genome. All four proteins share less than 40% sequence identity with each other and also with the identified members of AKR superfamily and hence deserve to be assigned in new families. Dissimilarity in sequences is also reflected through their substrate specificity. While reduction of trans-2-nonenal, a LPO-derived reactive aldehyde was common across the four proteins, these proteins were found to be activated during heat, salt, Cd, As, and butachlor treatments, and their ectopic expression in Escherichia coli conferred tolerance to the above abiotic stresses. These findings affirm the role of AKRs in providing a broad tolerance to environmental stresses conceivably by detoxifying the stress-generated reactive aldehydes.     

Improved reactive aldehyde,salt and cadmium tolerance of transgenic barley due to the expression of aldo–keto reductase genes

Under various stress conditions, plant cells are exposed to oxidative damage which triggers lipid peroxidation. Lipid peroxide breakdown products include protein crosslinking reactive aldehydes. These are highly damaging to living cells. Stress-protective aldo–keto reductase (AKR) enzymes are able to recognise and modify these molecules, reducing their toxicity. AKRs not only modify reactive aldehydes but may synthesize osmoprotective sugar alcohols as well. The role of these mixed function enzymes was investigated under direct reactive aldehyde, heavy metal and salt stress conditions. Transgenic barley (Hordeum vulgare L.) plants constitutively expressing AKR enzymes derived from either thale cress (Arabidopsis thaliana) (AKR4C9) or alfalfa (Medicago sativa) (MsALR) were studied. Not only AKR4C9 but MsALR expressing plants were also found to produce more sorbitol than the non-transgenic (WT) barley. Salinity tolerance of genetically modified (GM) plants improved, presumably as a consequence of the enhanced sorbitol content. The MsALR enzyme expressing line (called 51) exhibited almost no symptoms of salt stress. Furthermore, both transgenes were shown to increase reactive aldehyde (glutaraldehyde) tolerance. Transgenic plants also exhibited better cadmium tolerance compared to WT, which was considered to be an effect of the reduction of reactive aldehyde molecules. Transgenic barley expressing either thale cress or alfalfa derived enzyme showed improved heavy metal and salt tolerance. Both can be explained by higher detoxifying and sugar alcohol producing activity. Based on the presented data, we consider AKRs as very effective stress-protective enzymes and their genes provide promising tools in the improvement of crops through gene technology.     

A novel source of methylglyoxal and glyoxal in retina: implications for age-related macular degeneration

Aging of retinal pigment epithelial (RPE) cells of the eye is marked by accumulations of bisretinoid fluorophores; two of the compounds within this lipofuscin mixture are A2E and all-trans-retinal dimer. These pigments are implicated in pathological mechanisms involved in some vision-threatening disorders including age-related macular degeneration (AMD). Studies have shown that bisretinoids are photosensitive compounds that undergo photooxidation and photodegradation when irradiated with short wavelength visible light. Utilizing ultra performance liquid chromatography (UPLC) with electrospray ionization mass spectrometry (ESI-MS) we demonstrate that photodegradation of A2E and all-trans-retinal dimer generates the dicarbonyls glyoxal (GO) and methylglyoxal (MG), that are known to modify proteins by advanced glycation endproduct (AGE) formation. By extracellular trapping with aminoguanidine, we established that these oxo-aldehydes are released from irradiated A2E-containing RPE cells. Enzyme-linked immunosorbant assays (ELISA) revealed that the substrate underlying A2E-containing RPE was AGE-modified after irradiation. This AGE deposition was suppressed by prior treatment of the cells with aminoguanidine. AGE-modification causes structural and functional impairment of proteins. In chronic diseases such as diabetes and atherosclerosis, MG and GO modify proteins by non-enzymatic glycation and oxidation reactions. AGE-modified proteins are also components of drusen, the sub-RPE deposits that confer increased risk of AMD onset. These results indicate that photodegraded RPE bisretinoid is likely to be a previously unknown source of MG and GO in the eye.     

Transcriptome Analysis of the Hippocampus in Novel Rat Model of Febrile Seizures

Febrile seizures (FS) are the most common type of convulsive events in infants and young children, but the precise underlying genetic mechanism remains to be explored. To investigate the underlying pathogenic factors in FS and subsequent epilepsy, alterations in gene expression between the two new strains of rats (hyperthermia-prone [HP] vs hyperthermia-resistant [HR]), were investigated by using the Whole Rat Genome Oligo Microarray. This process identified 1,140 differentially expressed genes (DEGs; 602 upregulated and 538 downregulated), which were analyzed to determine significant Gene Ontology (GO) categories, signaling pathways and gene networks. Based on the GO analyses, the modified genes are closely related to various FS pathogenesis factors, including immune and inflammatory responses and ion transport. Certain DEGs identified have not been previously examined in relation to FS pathogenesis. Among these genes is dipeptidyl peptidase 4 (DPP4), a gene closely linked to interleukin 6 (IL-6), which played a key role in the gene network analysis. Furthermore, sitagliptin, a DPP4 inhibitor significantly decreased epileptic discharge in rats, observed via electroencephalogram, suggesting an important role for DPP4 in FS. The effectiveness of sitagliptin in reducing seizure activity may occur through a mechanism that stabilizes cellular Ca²⁺ homeostasis. In addition, DPP4 expression may be regulated by DNA methylation. The hippocampal gene expression profiles in novel rat models of FS provides a large database of candidate genes and pathways, which will be useful for researchers interested in disorders of neuronal excitability.     

Proteomic Analysis of MG132-Treated Germinating Pollen Reveals Expression Signatures Associated with Proteasome Inhibition

Chemical inhibition of the proteasome has been previously found to effectively impair pollen germination and tube growth in vitro. However, the mediators of these effects at the molecular level are unknown. By performing 2DE proteomic analysis, 24 differentially expressed protein spots, representing 14 unique candidate proteins, were identified in the pollen of kiwifruit (Actinidia deliciosa) germinated in the presence of the MG132 proteasome inhibitor. qPCR analysis revealed that 11 of these proteins are not up-regulated at the mRNA level, but are most likely stabilized by proteasome inhibition. These differentially expressed proteins are predicted to function in various pathways including energy and lipid metabolism, cell wall synthesis, protein synthesis/degradation and stress responses. In line with this evidence, the MG132-induced changes in the proteome were accompanied by an increase in ATP and ROS content and by an alteration in fatty acid composition.     

GSEA-SNP identifies genes associated with Johne’s disease in cattle

SNP-based gene-set enrichment analysis from single nucleotide polymorphisms, or GSEA-SNP, is a tool to identify candidate genes based on enrichment analysis of sets of genes rather than single SNP associations. The objective of this study was to identify modest-effect genes associated with Mycobacterium avium subsp. paratuberculosis (Map) tissue infection or fecal shedding using GSEA-SNP applied to KEGG pathways or Gene Ontology (GO) gene sets. The Illumina Bovine SNP50 BeadChip was used to genotype 209 Holstein cows for the GSEA-SNP analyses. For each of 13,744 annotated genes genome-wide located within 50 kb of a Bovine SNP50 SNP, the single SNP with the highest Cochran-Armitage Max statistic was used as a proxy statistic for that gene’s strength of affiliation with Map. Gene-set enrichment was tested using a weighted Kolmogorov-Smirnov-like running sum statistic with data permutation to adjust for multiple testing. For tissue infection and fecal shedding, no gene sets in KEGG pathways or in GO sets for molecular function or cellular component were enriched for signal. The GO biological process gene set for positive regulation of cell motion (GO:0051272, q = 0.039, 5/11 genes contributing to the core enrichment) was enriched for Map tissue infection, while no GO biological process gene sets were enriched for fecal shedding. GSEA-SNP complements traditional SNP association approaches to identify genes of modest effects as well as genes with larger effects as demonstrated by the identification of one locus that we previously found to be associated with Map tissue infection using a SNP-by-SNP genome-wide association study.     

Comparative analysis of protein interaction networks reveals that conserved pathways are susceptible to HIV-1 interception

Background

Human immunodeficiency virus type one (HIV-1) is the major pathogen that causes the acquired immune deficiency syndrome (AIDS). With the availability of large-scale protein-protein interaction (PPI) measurements, comparative network analysis can provide a promising way to study the host-virus interactions and their functional significance in the pathogenesis of AIDS. Until now, there have been a large number of HIV studies based on various animal models. In this paper, we present a novel framework for studying the host-HIV interactions through comparative network analysis across different species.

Results

Based on the proposed framework, we test our hypothesis that HIV-1 attacks essential biological pathways that are conserved across species. We selected the Homo sapiens and Mus musculus PPI networks with the largest coverage among the PPI networks that are available from public databases. By using a local network alignment algorithm based on hidden Markov models (HMMs), we first identified the pathways that are conserved in both networks. Next, we analyzed the HIV-1 susceptibility of these pathways, in comparison with random pathways in the human PPI network. Our analysis shows that the conserved pathways have a significantly higher probability of being intercepted by HIV-1. Furthermore, Gene Ontology (GO) enrichment analysis shows that most of the enriched GO terms are related to signal transduction, which has been conjectured to be one of the major mechanisms targeted by HIV-1 for the takeover of the host cell.

Conclusions

This proof-of-concept study clearly shows that the comparative analysis of PPI networks across different species can provide important insights into the host-HIV interactions and the detailed mechanisms of HIV-1. We expect that comparative multiple network analysis of various species that have different levels of susceptibility to similar lentiviruses may provide a very effective framework for generating novel, and experimentally verifiable hypotheses on the mechanisms of HIV-1. We believe that the proposed framework has the potential to expedite the elucidation of the important mechanisms of HIV-1, and ultimately, the discovery of novel anti-HIV drugs.

     

Characterization of Two Novel Aldo-Keto Reductases from Arabidopsis: Expression Patterns, Broad Substrate Specificity, and an Open Active-Site Structure Suggest a Role in Toxicant Metabolism Following Stress

Aldo-keto reductases (AKRs) are widely distributed in nature and play numerous roles in the metabolism of steroids, sugars, and other carbonyls. They have also frequently been implicated in the metabolism of exogenous and endogenous toxicants, including those stimulated by stress. Although the Arabidopsis genome includes at least 21 genes with the AKR signature, very little is known of their functions. In this study, we have screened the Arabidopsis thaliana genomic sequence for genes with significant homology to members of the mammalian AKR1 family and identified four homologues for further study. Following alignment of the predicted protein sequences with representatives from the AKR superfamily, the proteins were ascribed not to the AKR1 family but to the AKR4C subfamily, with the individual designations of AKR4C8, AKR4C9, AKR4C10, and AKR4C11. Expression of two of the genes, AKR4C8 and AKR4C9, has been shown to be coordinately regulated and markedly induced by various forms of stress. The genes have been overexpressed in bacteria, and recombinant proteins have been purified and crystallized. Both enzymes display NADPH-dependent reduction of carbonyl compounds, typical of the superfamily, but will accept a very wide range of substrates, reducing a range of steroids, sugars, and aliphatic and aromatic aldehydes/ketones, although there are distinct differences between the two enzymes. We have obtained high-resolution crystal structures of AKR4C8 (1.4 Å) and AKR4C9 (1.25 Å) in ternary complexes with NADP⁺ and acetate. Three extended loops, present in all AKRs and responsible for defining the cofactor- and substrate-binding sites, are shorter in the 4C subfamily compared to other AKRs. Consequently, the crystal structures reveal open and accommodative substrate-binding sites, which correlates with their broad substrate specificity. It is suggested that the primary role of these enzymes may be to detoxify a range of toxic aldehydes and ketones produced during stress, although the precise nature of the principal natural substrates remains to be determined.     

Quantitative Evaluation of Aldo-keto Reductase Expression in Hepatocellular Carcinoma (HCC) Cell Lines

The involvement of aldo–keto reductases (AKRs) in tumorigenesis is widely reported, but their roles in the pathological process are not generally recognized due to inconsistent measurements of their expression. To overcome this problem, we simultaneously employed real-time PCR to examine gene expression and multiple reaction monitoring (MRM) of mass spectrometry (MS) to examine the protein expression of AKRs in five different hepatic cell lines. These include one relatively normal hepatic cell line, L-02, and four hepatocellular carcinoma (HCC) cell lines, HepG2, HuH7, BEL7402 and SMMC7721. The results of real-time PCR showed that expression of genes encoding the AKR1C family members rather than AKR1A and AKR1B was associated with tumor, and most of genes encoding AKRs were highly expressed in HuH7. Similar observations were obtained through MRM. Different from HuH7, the protein abundance of AKR1A and AKR1B was relatively consistent among the other four hepatic cell lines, while protein expression of AKR1C varied significantly compared to L-02. Therefore, we conclude that the abundant distribution of AKR1C proteins is likely to be associated with liver tumorigenesis, and the AKR expression status in HuH7 is completely different from other liver cancer cell lines. This study, for the first time, provided both overall and quantitative information regarding the expression of AKRs at both mRNA and protein levels in hepatic cell lines. Our observations put the previous use of AKRs as a biomarker into question since it is only consistent with our data from HuH7. Furthermore, the data presented herein demonstrated that quantitative evaluation and comparisons within a protein family at both mRNA and protein levels were feasible using current techniques.     

Malachite green induces cardiovascular defects in developing zebrafish (Danio rerio) embryos by blocking VEGFR-2 signaling

Malachite green (MG) is a triphenyl methane dye used in various fields that demonstrates high toxicity to bacteria and mammalian cells. When bud stage zebrafish embryos were treated with MG at 125, 150, and 175 ppb for 14 h, the development of trunk including intersomitic vessels was inhibited in MG-treated flk-1-GFP transgenic embyos. MG clearly induced whole growth retardation. MG induced severe cell death in trunk intersomite region of zebrafish embryos and in human vascular endothelial cells in a dose-dependent manner. MG inhibited heart rates and cardiac looping. MG attenuated whole blood formation and inhibited vascular endothelial growth factor (VEGF)-induced receptor (R)-2 phosphorylation in vascular endothelial cells. In conclusion, MG significantly alters the cardiovascular development causing growth retardation in zebrafish through the blocking VEGFR-2 activation in early cardiovascular development. It suggests that MG may be an environmental toxic agent with the potential to induce embryonic cardiovascular defects in vertebrates.     

Methylglyoxal detoxification by a DJ-1 family protein provides dual abiotic and biotic stress tolerance in transgenic plants

Methylglyoxal (MG) is a key signaling molecule resulting from glycolysis and other metabolic pathways. During abiotic stress, MG levels accumulate to toxic levels in affected cells. However, MG is routinely detoxified through the action of DJ1/PARK7/Hsp31 proteins that are highly conserved across kingdoms and mutations in such genes are associated with neurodegenerative diseases. Here, we report for the first time that, similar to abiotic stresses, MG levels increase during biotic stresses in plants, likely contributing to enhanced susceptibility to a wide range of stresses. We show that overexpression of yeast Heat shock protein 31 (Hsp31), a DJ-1 homolog with robust MG detoxifying capabilities, confers dual biotic and abiotic stress tolerance in model plant Nicotiana tabacum. Strikingly, overexpression of Hsp31 in tobacco imparts robust stress tolerance against diverse biotic stress inducers such as viruses, bacteria and fungi, in addition to tolerance against a range of abiotic stress inducers. During stress, Hsp31 was targeted to mitochondria and induced expression of key stress-related genes. These results indicate that Hsp31 is a novel attractive tool to engineer plants against both biotic and abiotic stresses.