Stress Response Regulons Research Articles

Redox-enabled electronic interrogation and feedback control of hierarchical and networked biological systems

Microelectronic devices can directly communicate with biology, as electronic information can be transmitted via redox reactions within biological systems. By engineering biology’s native redox networks, we enable electronic interrogation and control of biological systems at several hierarchical levels: proteins, cells, and cell consortia. First, electro-biofabrication facilitates on-device biological component assembly. Then, electrode-actuated redox data transmission and redox-linked synthetic biology allows programming of enzyme activity and closed-loop electrogenetic control of cellular function. Specifically, horseradish peroxidase is assembled onto interdigitated electrodes where electrode-generated hydrogen peroxide controls its activity. E. coli’s stress response regulon, oxyRS, is rewired to enable algorithm-based feedback control of gene expression, including an eCRISPR module that switches cell-cell quorum sensing communication from one autoinducer to another—creating an electronically controlled ‘bilingual’ cell. Then, these disparate redox-guided devices are wirelessly connected, enabling real-time communication and user-based control. We suggest these methodologies will help us to better understand and develop sophisticated control for biology.

The Virulence and Infectivity of Listeria monocytogenes Are Not Substantially Altered by Elevated SigB Activity.

Listeria monocytogenes is a bacterial pathogen capable of causing severe infections but also thriving outside the host. To respond to different stress conditions, L. monocytogenes mainly utilizes the general stress response regulon, which largely is controlled by the alternative sigma factor Sigma B (SigB). In addition, SigB is important for virulence gene expression and infectivity. Upon encountering stress, a large multicomponent protein complex known as the stressosome becomes activated, ultimately leading to SigB activation. RsbX is a protein needed to reset a "stressed" stressosome and prevent unnecessary SigB activation in nonstressed conditions. Consequently, absence of RsbX leads to constitutive activation of SigB even without prevailing stress stimulus. To further examine the involvement of SigB in the virulence of this pathogen, we investigated whether a strain with constitutively active SigB would be affected in virulence factor expression and/or infectivity in cultured cells and in a chicken embryo infection model. Our results suggest that increased SigB activity does not substantially alter virulence gene expression compared with the wild-type (WT) strain at transcript and protein levels. Bacteria lacking RsbX were taken up by phagocytic and nonphagocytic cells at a similar frequency to WT bacteria, both in stressed and nonstressed conditions. Finally, the absence of RsbX only marginally affected the ability of bacteria to infect chicken embryos. Our results suggest only a minor role of RsbX in controlling virulence factor expression and infectivity under these conditions.

A Systematic Quantitative Determination of the Antimicrobial Efficacy of Grape Seed Extract against Foodborne Bacterial Pathogens

Concerns regarding the role of antimicrobial resistance (AMR) in disease outbreaks are growing due to the excessive use of antibiotics. Moreover, consumers are demanding food products that are minimally processed and produced in a sustainable way, without the use of chemical preservatives or antibiotics. Grape seed extract (GSE) is isolated from wine industry waste and is an interesting source of natural antimicrobials, especially when aiming to increase sustainable processing. The aim of this study was to obtain a systematic understanding of the microbial inactivation efficacy/potential of GSE against Listeria monocytogenes (Gram-positive), Escherichia coli and Salmonella Typhimurium (Gram-negative) in an in vitro model system. More specifically, for L. monocytogenes, the effects of the initial inoculum concentration, bacterial growth phase and absence of the environmental stress response regulon (SigB) on the GSE microbial inactivation potential were investigated. In general, GSE was found to be highly effective at inactivating L. monocytogenes, with higher inactivation achieved for higher GSE concentrations and lower initial inoculum levels. Generally, stationary phase cells were more resistant/tolerant to GSE as compared to exponential phase cells (for the same inoculum level). Additionally, SigB appears to play an important role in the resistance of L. monocytogenes to GSE. The Gram-negative bacteria under study (E. coli and S. Typhimurium) were less susceptible to GSE as compared to L. monocytogenes. Our findings provide a quantitative and mechanistic understanding of the impact of GSE on the microbial dynamics of foodborne pathogens, assisting in the more systematic design of natural antimicrobial-based strategies for sustainable food safety.

The stressosome is required to transduce low pH signals leading to increased transcription of the amino acid-based acid tolerance mechanisms in Listeria monocytogenes.

Increasing proton concentration in the environment represents a potentially lethal stress for single-celled microorganisms. To survive in an acidifying environment, the foodborne pathogen Listeria monocytogenes quickly activates the alternative sigma factor B (σB), resulting in upregulation of the general stress response (GSR) regulon. Activation of σB is regulated by the stressosome, a multi-protein sensory complex involved in stress detection and signal transduction. In this study, we used L. monocytogenes strains harbouring two stressosome mutants to investigate the role of this complex in triggering expression of known amino acid-based resistance mechanisms in response to low pH. We found that expression of glutamate decarboxylase (gadD3) and arginine and agmatine deiminases (arcA and aguA1, respectively) were upregulated upon acid shock (pH 5 for 15 min) in a stressosome-dependent manner. In contrast, transcription of the arg operons (argGH and argCJBDF), which encode enzymes for the l-arginine biosynthesis pathway, were upregulated upon acid shock in a stressosome-independent manner. Finally, we found that transcription of argR, which encodes a transcriptional regulator of the arc and arg operons, was largely unaffected by acidic shock. Thus, our findings suggest that the stressosome plays a role in activating amino acid-based pH homeostatic mechanisms in L. monocytogenes . Additionally, we show that genes encoding the l-arginine biosynthesis pathway are highly upregulated under acidic conditions, suggesting that intracellular arginine can help withstand environmental acidification in this pathogen.

Parsed synthesis of pyocyanin via co-culture enables context-dependent intercellular redox communication

BackgroundMicrobial co-cultures and consortia are of interest in cell-based molecular production and even as “smart” therapeutics in that one can take advantage of division of labor and specialization to expand both the range of available functions and mechanisms for control. The development of tools that enable coordination and modulation of consortia will be crucial for future application of multi-population cultures. In particular, these systems would benefit from an expanded toolset that enables orthogonal inter-strain communication.ResultsWe created a co-culture for the synthesis of a redox-active phenazine signaling molecule, pyocyanin (PYO), by dividing its synthesis into the generation of its intermediate, phenazine carboxylic acid (PCA) from the first strain, followed by consumption of PCA and generation of PYO in a second strain. Interestingly, both PCA and PYO can be used to actuate gene expression in cells engineered with the soxRS oxidative stress regulon, although importantly this signaling activity was found to depend on growth media. That is, like other signaling motifs in bacterial systems, the signaling activity is context dependent. We then used this co-culture’s phenazine signals in a tri-culture to modulate gene expression and production of three model products: quorum sensing molecule autoinducer-1 and two fluorescent marker proteins, eGFP and DsRed. We also showed how these redox-based signals could be intermingled with other quorum-sensing (QS) signals which are more commonly used in synthetic biology, to control complex behaviors. To provide control over product synthesis in the tri-cultures, we also showed how a QS-induced growth control module could guide metabolic flux in one population and at the same time guide overall tri-culture function. Specifically, we showed that phenazine signal recognition, enabled through the oxidative stress response regulon soxRS, was dependent on media composition such that signal propagation within our parsed synthetic system could guide different desired outcomes based on the prevailing environment. In doing so, we expanded the range of signaling molecules available for coordination and the modes by which they can be utilized to influence overall function of a multi-population culture.ConclusionsOur results show that redox-based signaling can be intermingled with other quorum sensing signaling in ways that enable user-defined control of microbial consortia yielding various outcomes defined by culture medium. Further, we demonstrated the utility of our previously designed growth control module in influencing signal propagation and metabolic activity is unimpeded by orthogonal redox-based signaling. By exploring novel multi-modal strategies for guiding communication and consortia outcome, the concepts introduced here may prove to be useful for coordination of multiple populations within complex microbial systems.

Novel RclSAR three-component system regulates expression of the intI1 gene in the stationary growth phase

The rapid and appropriate response of Pseudomonas spp. to environmental fluctuations has been enabled by numerous signal transduction regulatory systems. Regulatory systems in Pseudomonas aeruginosa are organized in a complex network which provides quick and fine-tuned cellular response through regulation of virulence and antibiotic resistance determinants production. Mobile integrons represent genetic elements included in the rapid dissemination of multiple antibiotic resistance determinants. The key factor of integron dynamics is enzyme integrase. So far, global regulators LexA, RpoS and PsrA have been recognized as regulators of the intI1 transcription. In this study, we discovered novel activator of the intI1 transcription, sensor kinase RclS, in Pseudomonas putida WCS358. This regulation is limited to stationary growth phase and appears to be indirect, at least through regulation of the rpoS expression. Sensor kinase RclS is a part of novel three-component system Rcl (Roc-like) together with two response regulators, RclR and RclA. RclS acted as a negative regulator of the rclA transcription, while the role in the rclR transcription regulation could not be defined. The RclSAR regulatory system seems to be a part of complex intI1 regulatory network which includes major stress response (SOS and RpoS) regulons.

Functional analysis of colonization factor antigen I positive enterotoxigenic Escherichia coli identifies genes implicated in survival in water and host colonization

Enterotoxigenic Escherichia coli (ETEC) expressing the colonization pili CFA/I are common causes of diarrhoeal infections in humans. Here, we use a combination of transposon mutagenesis and transcriptomic analysis to identify genes and pathways that contribute to ETEC persistence in water environments and colonization of a mammalian host. ETEC persisting in water exhibit a distinct RNA expression profile from those growing in richer media. Multiple pathways were identified that contribute to water survival, including lipopolysaccharide biosynthesis and stress response regulons. The analysis also indicated that ETEC growing in vivo in mice encounter a bottleneck driving down the diversity of colonizing ETEC populations.

A redox-based electrogenetic CRISPR system to connect with and control biological information networks

Electronic information can be transmitted to cells directly from microelectronics via electrode-activated redox mediators. These transmissions are decoded by redox-responsive promoters which enable user-specified control over biological function. Here, we build on this redox communication modality by establishing an electronic eCRISPR conduit of information exchange. This system acts as a biological signal processor, amplifying signal reception and filtering biological noise. We electronically amplify bacterial quorum sensing (QS) signaling by activating LasI, the autoinducer-1 synthase. Similarly, we filter out unintended noise by inhibiting the native SoxRS-mediated oxidative stress response regulon. We then construct an eCRISPR based redox conduit in both E. coli and Salmonella enterica. Finally, we display eCRISPR based information processing that allows transmission of spatiotemporal redox commands which are then decoded by gelatin-encapsulated E. coli. We anticipate that redox communication channels will enable biohybrid microelectronic devices that could transform our abilities to electronically interpret and control biological function.

Small RNA ArcZ Regulates Oxidative Stress Response Genes and Regulons in Erwinia amylovora.

Erwinia amylovora, causative agent of fire blight disease of apple and pear trees, has evolved to use small RNAs for post-transcriptional regulation of virulence traits important for disease development. The sRNA ArcZ regulates several virulence traits, and to better understand its roles, we conducted a transcriptomic comparison of wild-type and ΔarcZ mutant E. amylovora. We found that ArcZ regulates multiple cellular processes including genes encoding enzymes involved in mitigating the threat of reactive oxygen species (katA, tpx, osmC), and that the ΔarcZ mutant has reduced catalase activity and is more susceptible to exogenous hydrogen peroxide. We quantified hydrogen peroxide production by apple leaves inoculated with E. amylovora and found that the while wild-type E. amylovora cells produce enough catalase to cope with defense peroxide, the ΔarcZ mutant is likely limited in virulence because of inability to cope with peroxide levels in host leaves. We further found that the ArcZ regulon overlaps significantly with the regulons of transcription factors involved in oxidative sensing including Fnr and ArcA. In addition, we show that ArcZ regulates arcA at the post-transcriptional level suggesting a role for this system in mediating adaptations to oxidative state, especially during disease development.

Genetic screens reveal novel major and minor players in magnesium homeostasis of Staphylococcus aureus.

Magnesium is one of the most abundant metal ions in living cells. Very specific and devoted transporters have evolved for transporting Mg2+ ions across the membrane and maintain magnesium homeostasis. Using genetic screens, we were able to identify the main players in magnesium homeostasis in the opportunistic pathogen Staphylococcus aureus. Here, we show that import of magnesium relies on the redundant activity of either CorA2 or MgtE since in absence of these two importers, bacteria require increased amounts of magnesium in the medium. A third CorA-like importer seems to play a minor role, at least under laboratory conditions. For export of magnesium, we identified two proteins, MpfA and MpfB. MpfA, is the main actor since it is essential for growth in high magnesium concentrations. We show that gain of function mutations or overexpression of the minor factor, MpfB, which is part of a sigmaB controlled stress response regulon, can compensate for the absence of MpfA.

One Extra Copy of lon Gene Causes a Dramatic Increase in Actinorhodin Production by Streptomyces coelicolor A3(2).

ATP-dependent Lon protease plays important roles in different physiological processes, including cellular differentiation of the bacteria and is a part of an important stress response regulon (HspR/HAIR). In Streptomyces, biosynthesis of secondary metabolites starts with cellular differentiation and stress is one of the factor that affect metabolite production. To clarify the effect of Lon protease on secondary metabolite production, we constructed a recombinant strain of Streptomyces coelicolor A3(2) that has one extra copy of lon gene with its own promoter and transcriptional terminator in its genome. Expression of lon gene in the recombinant strain was determined by quantitative real time (RT-qPCR). Actinorhodin and undecylprodigiosin production of the recombinant cell was measured in liquid R2YE and it was found to produce about 34 times more actinorhodin and 9 times more undecylprodigiosin than the wild-type at 168h of growth. Development of stable Streptomyces strains capable of producing high amounts of secondary metabolites is valuable for biotechnology industry. One extra copy of lon gene is enough to boost antibiotic production by S. coelicolor A3(2) and this change do not cause any metabolic burden in the cell.

Refining the Pneumococcal Competence Regulon by RNA Sequencing.

Competence for genetic transformation allows the opportunistic human pathogen Streptococcus pneumoniae to take up exogenous DNA for incorporation into its own genome. This ability may account for the extraordinary genomic plasticity of this bacterium, leading to antigenic variation, vaccine escape, and the spread of antibiotic resistance. The competence system has been thoroughly studied, and its regulation is well understood. Additionally, over the last decade, several stress factors have been shown to trigger the competent state, leading to the activation of several stress response regulons. The arrival of next-generation sequencing techniques allowed us to update the competence regulon, the latest report on which still depended on DNA microarray technology. Enabled by the availability of an up-to-date genome annotation, including transcript boundaries, we assayed time-dependent expression of all annotated features in response to competence induction, were able to identify the affected promoters, and produced a more complete overview of the various regulons activated during the competence state. We show that 4% of all annotated genes are under direct control of competence regulators ComE and ComX, while the expression of a total of up to 17% of all genes is affected, either directly or indirectly. Among the affected genes are various small RNAs with an as-yet-unknown function. Besides the ComE and ComX regulons, we were also able to refine the CiaR, VraR (LiaR), and BlpR regulons, underlining the strength of combining transcriptome sequencing (RNA-seq) with a well-annotated genome.IMPORTANCEStreptococcus pneumoniae is an opportunistic human pathogen responsible for over a million deaths every year. Although both vaccination programs and antibiotic therapies have been effective in prevention and treatment of pneumococcal infections, respectively, the sustainability of these solutions is uncertain. The pneumococcal genome is highly flexible, leading to vaccine escape and antibiotic resistance. This flexibility is predominantly facilitated by competence, a state allowing the cell to take up and integrate exogenous DNA. Thus, it is essential to obtain a detailed overview of gene expression during competence. This is stressed by the fact that administration of several classes of antibiotics can lead to competence. Previous studies on the competence regulon were performed with microarray technology and were limited to an incomplete set of known genes. Using RNA sequencing combined with an up-to-date genome annotation, we provide an updated overview of competence-regulated genes.

PsrA Regulator Connects Cell Physiology and Class 1 Integron Integrase Gene Expression Through the Regulation of lexA Gene Expression in Pseudomonas spp.

Pseudomonas aeruginosa, which is a clinically important representative of Pseudomonas spp., has been recognized as causative agent of severe nosocomial infections worldwide. An increase in antibiotic resistance of P. aeruginosa clinical strains could be attributed to their capacity to acquire resistance through mobile genetic elements such as mobile integrons that are present in one-half of multidrug-resistant P. aeruginosa strains. Mobile class 1 integrons are recognized as genetic elements involved in the rapid dissemination of multiple genes encoding for antibiotic resistance. The LexA protein is a major repressor of integrase transcription, but differences in transcription regulation among bacterial species have also been noted. In this study, the promoter activity of class 1 integron integrase gene (intI1) and its variant lacking the LexA binding site in Pseudomonas putida WCS358 wild type, ΔrpoS and ΔpsrA was analysed. The results show that the activity of the intI1 gene promoter decreased in the rpoS and psrA mutants in the stationary phase of growth compared to the wild type, which indicates the role of RpoS and PsrA proteins in the positive regulation of integrase transcription. Additionally, it was determined that the activity of the lexA gene promoter decreased in ΔrpoS and ΔpsrA, and thus, we propose that PsrA indirectly regulates the intI1 gene promoter activity through regulation of lexA gene expression in co-operation with some additional regulators. In this study, intI1 gene expression was shown to be controlled by two major stress response (SOS and RpoS) regulons, which indicates that integrase has evolved to use both systems to sense the cell status.

Spx, the central regulator of the heat and oxidative stress response in B. subtilis, can repress transcription of translation-related genes.

Spx is a Bacillus subtilis transcription factor that interacts with the alpha subunits of RNA polymerase. It can activate the thiol stress response regulon and interfere with the activation of many developmental processes. Here, we show that Spx is a central player orchestrating the heat shock response by up-regulating relevant stress response genes as revealed by comparative transcriptomic experiments. Moreover, these experiments revealed the potential of Spx to inhibit transcription of translation-related genes. By in vivo and in vitro experiments, we confirmed that Spx can inhibit transcription from rRNA. This inhibition depended mostly on UP elements and the alpha subunits of RNA polymerase. However, the concurrent up-regulation activity of stress genes by Spx, but not the inhibition of translation related genes, was essential for mediating stress response and antibiotic tolerance under the applied stress conditions. The observed inhibitory activity might be compensated in vivo by additional stress response processes interfering with translation. Nevertheless, the impact of Spx on limiting translation becomes apparent under conditions with high cellular Spx levels. Interestingly, we observed a subpopulation of stationary phase cells that contains raised Spx levels, which may contribute to growth inhibition and a persister-like behaviour of this subpopulation during outgrowth.

Antibiotic-Induced Genetic Variation: How It Arises and How It Can Be Prevented.

By targeting essential cellular processes, antibiotics provoke metabolic perturbations and induce stress responses and genetic variation in bacteria. Here we review current knowledge of the mechanisms by which these molecules generate genetic instability. They include production of reactive oxygen species, as well as induction of the stress response regulons, which lead to enhancement of mutation and recombination rates and modulation of horizontal gene transfer. All these phenomena influence the evolution and spread of antibiotic resistance. The use of strategies to stop or decrease the generation of resistant variants is also discussed.

A novel SigB(Q225P) mutation in Staphylococcus aureus retains virulence but promotes biofilm formation

Staphylococcus aureus is an important pathogen that produces abundant virulence factors, which cause various diseases that burden human health worldwide. The stress response regulon called sigma factor B (SigB) is a well-characterized global regulator that is involved in the regulation of S. aureus virulence, pigmentation, and biofilm formation. However, the regulatory network upon SigB in S. aureus is incompletely described. Here, we identified a novel substitution mutation, SigB(Q225P), which contributed the nonpigmented phenotype of S. aureus. The S. aureus mutant carrying SigB(Q225P) substitution lacks staphyloxanthin, a key virulence factor in protecting bacteria from host-oxidant killing, but retains bacterial pathogenicity with pleiotropic alterations in virulence factors, resulting in similar lethality and abscess formation ability in animal models. We also reported the SigB(Q225P) promotion of biofilm formation in S. aureus. Real-time quantitative polymerase chain reaction (RT-qPCR) revealed that the expression of nuc gene, which encodes thermonuclease, was significantly downregulated, resulting in accumulation of eDNA in the biofilm of SigB(Q225P) mutant strain. LacZ reporter assay showed that SigB(Q225P) influenced the activity of nuc promoter. Furthermore, electrophoretic mobility shift assay (EMSA) and Bio-layer interferometry (BLI) assay revealed that both SigB and SigB(Q225P) proteins could directly bind to nuc gene promoter; however, the binding activity decreased for SigB(Q225P). Our data renewed the understanding of the relationship between S. aureus golden pigment and its virulence and suggested that a single substitution mutation in SigB might enhance the biofilm formation of S. aureus by directly downregulating nuc expression.

Self-assembling dipeptide antibacterial nanostructures with membrane disrupting activity

Peptide-based supramolecular assemblies are a promising class of nanomaterials with important biomedical applications, specifically in drug delivery and tissue regeneration. However, the intrinsic antibacterial capabilities of these assemblies have been largely overlooked. The recent identification of common characteristics shared by antibacterial and self-assembling peptides provides a paradigm shift towards development of antibacterial agents. Here we present the antibacterial activity of self-assembled diphenylalanine, which emerges as the minimal model for antibacterial supramolecular polymers. The diphenylalanine nano-assemblies completely inhibit bacterial growth, trigger upregulation of stress-response regulons, induce substantial disruption to bacterial morphology, and cause membrane permeation and depolarization. We demonstrate the specificity of these membrane interactions and the development of antibacterial materials by integration of the peptide assemblies into tissue scaffolds. This study provides important insights into the significance of the interplay between self-assembly and antimicrobial activity and establishes innovative design principles toward the development of antimicrobial agents and materials.

The SOS and RpoS Regulons Contribute to Bacterial Cell Robustness to Genotoxic Stress by Synergistically Regulating DNA Polymerase Pol II.

Mitomycin C (MMC) is a genotoxic agent that induces DNA cross-links, DNA alkylation, and the production of reactive oxygen species (ROS). MMC induces the SOS response and RpoS regulons in Escherichia coli. SOS-encoded functions are required for DNA repair, whereas the RpoS regulon is typically induced by metabolic stresses that slow growth. Thus, induction of the RpoS regulon by MMC may be coincidental, because DNA damage slows growth; alternatively, the RpoS regulon may be an adaptive response contributing to cell survival. In this study, we show that the RpoS regulon is primarily induced by MMC-induced ROS production. We also show that RpoS regulon induction is required for the survival of MMC-treated growing cells. The major contributor to RpoS-dependent resistance to MMC treatment is DNA polymerase Pol II, which is encoded by the polB gene belonging to the SOS regulon. The observation that polB gene expression is controlled by the two major stress response regulons that are required to maximize survival and fitness further emphasizes the key role of this DNA polymerase as an important factor in genome stability.

Sequencing-relative to hybridization-based transcriptomics approaches better define Mycobacterium tuberculosis stress-response regulons

Mycobacterium tuberculosis (Mtb) infections cause tuberculosis (TB), an infectious disease which causes ∼1.5 million deaths annually. The ability of this pathogen to evade, escape and encounter immune surveillance is fueled by its adaptability. Thus, Mtb induces a transition in its transcriptome in response to environmental changes. Global transcriptome profiling has been key to our understanding of how Mtb responds to the different stress conditions it faces during its life cycle. While this was initially achieved using microarray technology, RNAseq is now widely employed. It is important to understand the correlation between the large amount of microarray based transcriptome data, which continues to shape our understanding of Mtb stress networks, and newer data being generated using RNAseq. We assessed how well the two platforms correlate using three well-defined stress conditions: diamide, hypoxia, and re-aeration. The data used here was generated by different individuals over time using distinct samples, providing a stringent test of platform correlation. While correlation between microarrays and sequencing was high upon diamide treatment, which causes a rapid reprogramming of the transcriptome, RNAseq allowed a better definition of the hypoxic response, characterized by subtle changes in the magnitude of gene-expression. RNAseq also allows for the best cross-platform reproducibility.

Molybdenum application enhances antioxidant enzyme activity and COR15a protein expression under cold stress in wheat

ABSTRACTUnder cold stress, reactive oxygen species (ROS) are considered the main source of damage to plant cells. Mechanisms of ROS scavenging in wheat are very important during stress and the antioxidant enzymes superoxide dismutase, Catalase and Glutathione peroxidase are key to facilitating ROS scavenging. Molybdenum (Mo) is involved in many plant physiological and biochemical processes including antioxidant enzymes. This study reports research to investigate the effect of Mo application in enhancing antioxidant enzymes in two wheat cultivars. The results confirmed that antioxidant defense is important in wheat that is exposed to abiotic stress and that changes in activities of antioxidant enzymes occurred during exposure of plants to low non-freezing temperatures and by adding Mo. Mo application had a positive effect on gene expression of both Cbf14 and COR15a protein expression, indicating upregulation of the stress response regulon. In addition, Mo enhanced antioxidant enzymes activity and improved frost tolerance.