In response to abiotic stresses, plants mount comprehensive stress-specific responses which mediate signal transduction cascades, transcription of relevant responsive genes and the accumulation of numerous different stress-specific transcripts and metabolites, as well as coordinated stress-specific biochemical and physiological readjustments. These natural mechanisms employed by plants are however not always sufficient to ensure plant survival under abiotic stress conditions. Biostimulants such as plant growth-promoting rhizobacteria (PGPR) formulation are emerging as novel strategies for improving crop quality, yield and resilience against adverse environmental conditions. However, to successfully formulate these microbial-based biostimulants and design efficient application programs, the understanding of molecular and physiological mechanisms that govern biostimulant-plant interactions is imperatively required. Systems biology approaches, such as metabolomics, can unravel insights on the complex network of plant-PGPR interactions allowing for the identification of molecular targets responsible for improved growth and crop quality. Thus, this review highlights the current models on plant defence responses to abiotic stresses, from perception to the activation of cellular and molecular events. It further highlights the current knowledge on the application of microbial biostimulants and the use of epigenetics and metabolomics approaches to elucidate mechanisms of action of microbial biostimulants. As sessile organisms, plants are constantly exposed to adverse environmental perturbations, such as abiotic stresses. Anthropogenic contributions and increasing climate change continuously exacerbate the detrimental effects of these stresses on crop productivity, thereby posing a threat to global food security. Evolutionarily, plants have developed a multi-layered, complex and highly regulated immune system and defences that involve sensing various danger signals and integration of this information to produce appropriate responses to diverse challenges, ensuring growth and development . These rPriming or pre-conditioning (of plant defences and adaptive mechanisms) as stress memory is a state in which plants are rendered more resistant to subsequent stresses, displaying faster and more efficient defence responses ,5,6. MulFurthermore, during stress encounters, metabolic perturbations are induced as part of the defence phenomenology, and some of these metabolic responses may persist following recovery, where the plant physiology returns to equilibrium ,20. ThisFurthermore, in addition to stress encounters, the plant (stress) memory or the priming phenomenology can be initiated by priming activators such as a natural or synthetic chemical compound or biostimulants. The latter are of interest for this review. The exposure of plants to these priming agents have shown to render the plant into the primed state, presenting thus opportunities for more effective use of plant priming in plant stress physiology studies and crop stress management . BiostimThus, this review, a critical synthesis of recent literature and data, provides a current knowledge base of plant responses to abiotic stresses, highlighting key models that explain the stress perception and signalling events as well as the cellular and molecular dynamics underlying plant responses to abiotic stresses. Furthermore, attention to the epigenetic and metabolomic changes, as underlying events in plant stress memory, is reviewed, offering opportunities within the context of systems biology approaches for the alleviation of abiotic stress and induction of priming. As mentioned above, this stress memory or priming can be activated by biostimulants. In the last decade, there has been exponentially growing attention towards biostimulants as a potential solution to mitigate the negative impacts of the changing climate on agriculture, and becoming one of the pillars of a new agricultural revolution for sustainable food production. However, there is a growing need for a sound scientific foundation to pave a theoretical framework that would contribute to the formulation of biostimulant products, with scientifically-based descriptions and credibility. To counteract the adverse effect of environmental perturbations, plants have evolved comprehensive defence mechanisms that help them tolerate abiotic stresses using physical adaptation as well as integrated molecular and cellular responses. The initial and crucial step in abiotic stress defence mechanisms is the perception of the stress signals and their transduction to activate the relevant adaptive molecular responses to ensure survival.Arabidopsis cyt) is maintained at nanomolar concentration levels via active transportation into the calcium stores or outside the cells into the apoplast, resulting in Ca2+ gradients of numerous magnitudes [2+] cyt influx and efflux patterns are evoked upon various stimuli perception as a response mechanism and this is controlled through different channels and pumps. These [Ca2+] cyt fluctuations by stimuli can occur in a repetitive manner in which the frequency and the amplitude of the signal are dependent on the type of the stimulus, thus making this a signature cyt and thus induce stomatal closure [During stress exposure, plants can amplify the initial stress signals, depending on the type of stress encountered and they do this by making use of phytohormones. These phytohormone-driven signalling can either trigger new signalling events that are similar to those of the initial signal or initiate an entirely different signalling event with different components . The mos closure . ABA is  closure , resulti closure . MAPK ca closure . These scis- and trans-element in the promoters of target genes and this type of transcriptional regulation is termed regulon [Plant defence responses, aiding in adaptation to abiotic stresses, are coordinated by networks of cellular and molecular mechanisms, fine-tuning changes in the growth and development of the plant. Significant progress has been made in elucidating these plant defences against environmental perturbations, which generally comprise alterations in the plant transcriptome, proteome and metabolome ,80,81,82 regulon . In thisNumerous regulons that are activated in response to abiotic stress have been identified in plants and are components of ABA, a principal phytohormone involved in the regulation of abiotic stress in plants by regulating an intricate gene regulatory system that permits plants to tolerate environmental perturbations . MyeloblRD29B, RAB18), cell cycle regulator genes (ICK1) and PP2Cs (ABI1 and ABI2)  class genes (nd ABI2) . RD22 any to ABA . In addiPlant growth and development regulation, together with the alleviation of detrimental effects of abiotic stresses, are crucial factors that determine the productivity of cultivated plants. Abiotic stresses are well known to negatively affect plant growth and development and are responsible for crop losses globally. However, the current knowledge on molecular and cellular mechanisms involved in mitigating these deleterious effects are still limited. Furthermore, biostimulants are increasingly being integrated into production systems, to modify underlying plant physiological and biochemical processes to enhance stress tolerance and productivity. By definition, biostimulants are diverse substances or microorganisms that are formulated to stimulate the plant’s natural processes to enhance plant productivity through different modes of action, irrespective of their nutrient content or  constituents ,92. AddiPlant biostimulants are available in a wide range of formulations with varying constituents but are generally categorised into four major groups based on their source and content. These groups include amino acid-containing products (AACP), hormone-containing products (HCP), humic substances (HS) and plant growth-containing microorganisms . AdditioSeveral possible key mechanisms of action induced by biostimulants in relation to abiotic stress alleviation have been elucidated and include ROS scavenging, membrane stability, osmoprotection, stomatal regulation, ion homeostasis, nutrient availability and metal chelation , howeverAzospirillum brasilense Sp245 lipopolysaccharides (LPS) stimulate growth (fresh weight and root length) in Arabidopsis thaliana [PGPR are increasingly being used as biostimulant formulations, showing potentials for improving plant health, development and sustainable increased yield. These soil bacteria that inhabit the rhizosphere interact symbiotically with the plant host to enhance plant growth. This chemical communication is translated into physiological benefits through various mechanisms which include nitrogen fixation, production of growth-stimulating phytohormones and solubilisation of mineral phosphates . A detaithaliana  and thisThe application of PGPR to induce abiotic stress tolerance in plants is increasingly being explored as an attractive strategy to regulate plant stress ,143,144 Under abiotic stresses PGPR employs different mechanisms to help plants survive the environmental changes including (i) production of ACC deaminase which lowers ET levels in plants ,146;  to the fifth carbon of the pyrimidine ring of cytosine nucleotide without altering the underlying DNA sequence . The modTEs make up a considerable proportion of the plant’s genome, therefore the regulation thereof is essential because they are potentially highly mutagenic and their accumulation limits survival . MoreovePopulus trichocarpa, an increase in methylation was observed under drought stress, with 10.04% compared to the watered plants with a 7.75% increase [Several examples have indicated that DNA methylation can either increase or decrease in response to stress and it appears that demethylation, which leads to gene expression activation, is a prompt response common in plants. In maize seedlings under cold stress exposure, hypomethylation was observed in root tissue following the expression of ZmMI1 and after seven days of cold exposure (recovery), the cold-induced decrease in methylation levels was not restored to basal levels . Additioincrease . Herein,increase . Methylaincrease . The accBoea hygrometrica genome which was consistent with the transcriptional changes in methylation pathway genes. As expected, the global promoter and gene body methylation levels were negatively correlated with the gene expression levels in both acclimated and dehydrated plants. Nonetheless, the promoter methylation variations in the CG and CHG contexts were significantly associated with the differential expression of genes required for fundamental genetic processes of DNA conformation, RNA splicing, translation and post-translational protein modification during acclimation, growth and rapid dehydration stress response. Additionally, the observed DNA methylation changes were associated with the upregulation of stress memory genes including pre-mRNA-splicing factor 38A, vacuolar amino acid transporter 1-like and UDP-sugar pyrophosphorylase, which may contribute to the enhancement of dehydration tolerance in B. hygrometrica plants. Furthermore, emerging studies have evidently pointed to DNA methylation as one of the key components of stress priming and memory ,216,217.In addition to alterations of DNA molecules through methylation or other processes, the chemical modification of histones is another main component of the epigenome. Histones are essential proteins involved in the packing and ordering of the DNA molecule into a fundamental structural unit of chromatin called a nucleosome. The latter consists of ~147 bp of DNA wrapped around a histone octamer that contains two copies each of H2A, H2B, H3 and H4, and accessibility of genomic DNA is regulated at this core particle . The N-tArabidopsis thaliana, a reference plant generally used for epigenetic studies in plant responses to abiotic stresses, under prolonged cold stress, an increase in H3K9 and H3K27 dimethylation and a decrease in H3K4 trimethylation was observed [A. thaliana that exhibit flowering delays in the first season [In the context of plant-environment interactions, studies have revealed that histone modifications that are closely related to plant responses to abiotic stress exposure are histone acetylation, de-acetylation, methylation and demethylation, which are catalysed by histone acetyltransferases (HATs), histone deacetylases (HDACs), histone methyltransferases (HMTs) and histone demethylases (HDMs), respectively ,226. Theobserved . Furthert season . Sokol et season , using wA. thaliana [Similarly, to DNA methylation, histone modifications as probable marks of priming and stress memory have been reported in several studies ,231,232.thaliana  and coulTarget gene repressions by small non-coding RNAs  have alsBrachypodium distachyon under normal plant growth conditions. The reported hypermethylation was correlated to an increase in the abundance of methyltransferases involved in the maintenance and regulation of DNA methylation. Additionally, the PGB induced hypermethylation levels remained constant when compared to the naïve plants after five and eight days of drought treatment, suggesting that Bacillus subtilis B26 potentially acts at an epigenetic level to increase drought stress tolerance in Brachypodium distachyon by inducing DNA methylation changes under normal conditions (priming) that, in turn, increase drought resistance by allowing the expression of drought-responsive genes. De Palma et al. [Trichoderma harzianum (a fungus used in biostimulant formulations) to induce epigenetic modifications, namely DNA methylation, providing an insight into plant-microbial interplay in tomato roots. The results showed a decrease in DNA methylation levels at the different time points, which correlated to transcriptional regulation of defence-related genes. The observed epiregulator expression mediates plant defence mechanisms by the interaction with Trichoderma and/or other Trichoderma-induced effects. Recent studies have extended our understanding of epigenetic modifications induced by microbial biostimulants; however, the majority of these studies mainly focused on epigenetic mechanisms associated with microbial biostimulants under biotic stress [Evidence on epigenetic modifications induced by microbial biostimulants has been reported by Gagné-Bourque et al.  in whicha et al.  very recc stress ,243,244 omics science that has been widely applied in different fields. This multidisciplinary omics science is defined as the comprehensive, qualitative and quantitative analysis of all the small molecules, termed metabolites, in a biological system [Various cellular and biochemical changes induced by abiotic stress are defined at the plant metabolism level. In a simplified description, under abiotic stresses, the plant metabolism is perturbed due to factors such as inhibition of enzymes or increased demand for compounds required for normal growth and development. Consequently, the plant’s metabolic network must continually readjust under these unfavourable conditions to maintain the normal metabolomic homeostasis and allow for the production of defence-related compounds that aid in stress tolerance. Accordingly, the current implementation of metabolomic approaches provides a thorough analysis of vital components of the plant’s defence responses to abiotic stress. Here, metabolomics is a powerful tool that provides an overview of how an organism’s metabolic network is regulated in response to stimuli. Metabolomics is a rapidly expanding l system ,246. The metabolome comprises a pool of low-molecular-weight metabolites usually less than 1500 Da. These metabolites are considered final products of cellular regulatory processes (gene expression and protein activity), modulating processes between the genome and the environment. In this regard, metabolomics is a cornerstone in the integration of the ‘-omics’ technologies that contribute to a systems biology overview . As suchomics’ science that has proven indispensable in interrogating cellular biochemistry and metabolism and has established itself as a powerful research tool to address biological questions related to plant-environment interactions [in toto [Metabolomics is a multidisciplinary ‘ractions ,250,251.ractions ,252. Owi[in toto . Consequ[in toto ,255, alo[in toto ,258,259.Metabolite profiling of plants growing under abiotic stress conditions has provided crucial information about changes at biochemical and molecular levels underlying plant growth and adaptation. It is worth mentioning that metabolomic changes that have been reported in plants subjected to stress conditions are dependent on different causes; thus, these metabolic alterations have different significance and are expected to differently correlate with stress tolerance. Metabolomic reprogramming due to adverse environmental conditions involves complex and highly regulated molecular events some of which include (1) the stability and catalytic activity of enzymes involved in the biosynthesis/degradation of particular metabolites, (2) the adjustment of metabolite concentrations to re-establish homeostasis and normal metabolic fluxes and (3) the accumulation of compounds involved in mediating stress tolerance mechanisms .Under abiotic stress conditions, the total number, concentration and types of metabolites are significantly altered. The alteration in gene expression is directly reflected in the metabolite profiles of plants. Acquiring knowledge about these differential metabolite profiles , and their modulation upon the onset of various abiotic stresses is highly fundamental. Reported metabolite changes have opened up the scope for the identification of viable metabolic markers which are important for abiotic stress tolerance of plants ,262,263.For example, upon exposure to abiotic stresses such as temperature, salinity and drought, plants accumulate a wide of compatible osmolytes which primarily function to maintain turgor. The accumulated solutes which vary among species include sugars , polyols, betaines and amino acids such as proline ,270. SomA. thaliana’s primary metabolism was found to be boosted through alterations of the tricarboxylic acids (TCA) namely citrate, fumarate, malate and oxoglutarate. Furthermore, the amplification of the phenylpropanoid biosynthesis and the octadecanoic pathway was also observed. Evidence on plant metabolic changes that occur as a result of priming have been reported and include the reprogramming of the primary metabolism and differential biosynthesis of secondary metabolites ,277,278,The metabolic reprogramming of the primary and secondary metabolism indicates that multiple metabolic pathways are involved in the priming phenomenon. The interconnectedness of these metabolic pathways has been reported to have feedback loops that allow for rapid activation of cellular defences to potential adverse conditions such as abiotic stress . DespiteAs highlighted in sections above, the priming phenomenology is described at different layers including the metabolome and epigenome which can be explorable avenues for the underlying priming effect of biostimulants to enhance plant protective machinery against environmental stresses. Plant priming, therefore, presents a promising alternative approach due to the long-term and broad-spectrum resistance it provides against abiotic stress, providing an effective mechanism for crop protection under abiotic stresses. Accordingly, a comprehensive and mechanistic understanding (at molecular and cellular levels) of the beneficial effects of the biostimulants involved in priming could pave the way to design novel strategies that will aid plants in adverse environmental conditions, thus contributing to sustainable food security. Biostimulants, such as plant growth-promoting rhizobacteria (PGPR)-based formulations, therefore represent potentials to provide sustainable and economically favourable solutions that could introduce novel approaches to improve agricultural practices and crop productivity. However, to effectively establish and devise novel biostimulant-based agricultural strategies, there is a necessity to firstly understand the physiology and biochemistry governing the interactions between biostimulants and plants at both cellular and molecular levels. This knowledge gap—decoding molecular and physiological mechanisms underlying biostimulant action (by exploring the epigenomic and metabolomic approaches)—is one of the main bottlenecks that hamper the biostimulant field and industries from implementing and maximising the value of  such formulations in agronomic practices. Climate change is depleting natural resources and exerting negative impacts on crop production, thus threatening food security. Modern agriculture is shifting toward improved crop yield and quality by exploiting the natural priming phenomenon in plants. In this context, the exploitation of biostimulants to enhance abiotic stress tolerance has increasingly become a strategy worth pursuing among the scientific community and agriculture industry. Biostimulants could pose as feasible alternatives in enhancing plant growth and increased stress tolerance. Numerous reports have elucidated the potential effects of biostimulant applications using various microorganisms such as PGPR; however, their comprehensive modes of action and underlying molecular mechanisms in relation to their priming effects and growth promotion are still enigmatic. This review discusses the growing literature and the current knowledge on plant defence responses to abiotic stresses and the integration of biostimulants in agricultural production systems, pointing out some knowledge gaps. The increasing use of biostimulants has demonstrated the positive effects of these formulations which include the modification of plant physiological and biochemical processes to enhance stress tolerance and productivity. This review further briefly highlights the application of metabolomics and epigenetic approaches in studying plant defence responses to abiotic stresses and defence priming.


Engagement in a wide array of mental, social, and physical leisure activities confers several health benefits. Indeed, theories of successful aging argue that an active lifestyle serves as an important criterion for maintaining high levels of psychological, functional, and physical well-being in old age. Findings from parallel studies also show that people who hold positive (self-)views of aging exhibit higher and maintained levels of well-being over time. Yet, whether views of aging enhances the link between activity engagement and well-being - and whether they do so on a daily basis – remains unknown. This study therefore sought to extend prior literature by examining the relationship between activity engagement, subjective age, and affective ratings within-person over several days. Old adults  in the Mindfulness and Anticipatory Coping Every Day (MACED) study completed an 8-day daily diary. Participants reported on their positive and negative affect, the age they subjectively felt compared to their actual age, and the number and types of leisure activities in which they engaged. Results from multilevel analyses indicate that people felt more positive on days when they also engaged in more activities  than usual. Moreover, the effect of activity engagement was most pronounced on days when people felt younger than usual. No effects were found for negative affect. Preliminary findings suggest that people benefit psychologically from daily leisure activities and a positive self-view of aging.


Although research on cancer biology in recent decades has unveiled the main genetic perturbations driving the onset of tumorigenesis, we are still far from properly treating this disease without the occurrence of drug resistance and metastatic burden. This achievement is hampered by the onset of intra-tumour heterogeneity (ITH), which increases cancer cell fitness and plasticity, thereby fostering cell adaptation to foreign environments and stimuli. In this review, we discuss the contribution of the epigenetic factors in sustaining ITH and their interplay with the tumour microenvironment. We also highlight the recent technological advancements that are contributing to defining the epigenetic mechanisms governing tumour heterogeneity at the single-cell level. Cancer is a group of heterogeneous diseases that results from the occurrence of genetic alterations combined with epigenetic changes and environmental stimuli that increase cancer cell plasticity. Indeed, multiple cancer cell populations coexist within the same tumour, favouring cancer progression and metastatic dissemination as well as drug resistance, thereby representing a major obstacle for treatment. Epigenetic changes contribute to the onset of intra-tumour heterogeneity (ITH) as they facilitate cell adaptation to perturbation of the tumour microenvironment. Despite being its central role, the intrinsic multi-layered and reversible epigenetic pattern limits the possibility to uniquely determine its contribution to ITH. In this review, we first describe the major epigenetic mechanisms involved in tumourigenesis and then discuss how single-cell-based approaches contribute to dissecting the key role of epigenetic changes in tumour heterogeneity. Furthermore, we highlight the importance of dissecting the interplay between genetics, epigenetics, and tumour microenvironments to decipher the molecular mechanisms governing tumour progression and drug resistance.  Cancer is characterized by a high degree of heterogeneity that occurs at both the inter- and intra-tumour levels . Inter-tITH arises from at least three mechanisms, which are not mutually exclusive and act together to create a complex system with multiple layers of heterogeneity: (1) genetic heterogeneity, due to stochastic accumulation of mutations that results in tumours acquiring subclones with distinct genotypes during tumour evolution; (2) non-genetic heterogeneity from variations in regulatory mechanisms, including epigenetic, posttranscriptional, and posttranslational modifications, among others; (3) tumour microenvironmental (TME) heterogeneity, due to selective pressures in distinct regions of the tumour . VaryingHeterogeneous tumour ecosystems have traditionally been profiled using bulk experimentation studies that only revealed an averaged cellular behaviour and thus have heavily underestimated the complex intra-tumour heterogeneity that characterizes cancer samples. With the advent of single-cell multi-omics technologies, cancer research has shifted to a new paradigm that has facilitated a comprehensive analysis of the various cellular programs within human tumours at single-cell resolution and thus improved our understanding of tumour heterogeneity and cancer evolution.In this review, we discuss the contribution of epigenetic alterations to tumour heterogeneity and their interplay with other sources of ITH. In addition, we provide an overview of the novel approaches that can be applied to dissect the complexity of the molecular mechanisms governing cancer-specific epigenetic changes at a single-cell level.In multicellular organisms, different cell types arise from the same cell of origin through a hierarchical differentiation process that specifies cell functions and tissue localisations. This specified, yet diversified tissue organization is disrupted in tumour cells, resulting in a heterogeneous phenotypic pattern. In 1976, Nowell proposed that cancer development is driven by clonal evolution, which results in adapted clones driving heterogeneity . One yeaThese findings indicate that the information retrieved from a single patient biopsy might not be sufficient to determine the optimal first-line treatment. This can result in incomplete tumour eradication and increased selective pressure for other cancer cell subpopulations, leading to increased aggressiveness and resistance of the remaining clones and subsequent failure of the second-line treatment.Over the last 30 years, an increasing amount of evidence has been gathered showing that cancer development was driven by not only genetic but also by non-genetic alterations linked to epigenetic dysregulation, including changes in the 3D genome organization, chromatin state, and DNA methylation. In comparison to the mutational load, which is considered a vertical transfer and involves cell division and the subsequent survival of the daughter cells, epigenetic changes are dynamic and reversible, allowing cancer cells to adapt to novel environmental stimuli, such as nutrient deprivation, oxidative stress, and microenvironment-related signals ,14. SincMultiple layers of epigenetic control can be corrupted during cancer progression and metastasis. By analyzing the different patterns of epigenetic alterations in multiple cancers, it has been shown that epigenetic heterogeneity represents a hallmark of cancer cells. Diverse routes drive towards epigenetic aberration during tumour progression and metastasis formation, giving rise to eITH, which increases cancer cell plasticity . HoweverThe first epigenetic marker extensively studied in the context of cancer was DNA methylation. DNA methylation occurs at cytosine-guanine sites, and in healthy tissues a large portion of the genome (60–80%) is methylated, ensuring genome stability and silencing of repetitive elements within the heterochromatic regions. However, in cancer cells, a large fraction of the genome actually shows a hypomethylated state compared to the healthy controls, with enrichment at repetitive elements (RE) ,21. HypoBesides the methylation of tumour suppressor genes, DNA methylation can also affect TF binding, as was shown for hypoxia-induced TF  and 3D gBesides DNA methylation, histone posttranslational modifications (PTM) play a central role in establishing different chromatin contexts that can modulate both genetic and non-genetic genome functions ,34. The cis, as it affects the methylation of the neighbouring H3.3K36 residue [On the other hand, mutations in histone genes can alter the epigenetic landscape by mimicking a certain histone PTM and make histones insensitive to other PTMs on the mutated locus. These mutations have been detected mainly in genes encoding histone H3 variants, such as a Lys-27-Met mutation in genes coding for histone 3.3 variants (H3K27M), identified in paediatric high-grade gliomas (HGG) . This mu residue . The ini residue . cis-regulatory elements hampers gene regulation by limiting the access of TFs and chromatin factors to the cognate DNA elements, thereby representing a major barrier for transcription to occur. Nucleosomal remodeling is mainly driven by multi-subunit complexes, termed chromatin remodeling complexes, composed of different protein families with the most prominent member in the context of cancer being the mSWI/SNF complex. These ATP-dependent remodeling factors disrupt the connection between nucleosomes and DNA by hydrolysis, allowing them to deposit, eject, or slide nucleosomes, thereby altering the chromatin structure and ultimately allowing for the establishment of an active chromatin state [Nucleosome occupancy at both distal and proximal in state . In >20%in state , resultiin state , linkingIn a hallmark study, Alonso-Curbelo and colleagues showed that injury in pancreatic tissue harbouring a Kras mutation drives the establishment of an epigenetic state more resembling pancreatic ductal adenocarcinoma (PDAC) than healthy pancreatic tissue . Interescis-regulatory elements, which ultimately result in an altered epigenetic program. Although the mechanisms and players that drive this epigenetic reprogramming are not yet completely understood, some players have already been identified and linked to cancer heterogeneity and tumour progression. Cancer cells can respond in different ways to environmental stimuli, which often only affect a fraction of the whole cancer bulk. In response to stimuli, master TFs are often deregulated, which in turn have a great impact on cancer cell phenotypes. It has been proposed that TFs play a central role in driving epigenetic reprogramming, by suppressing the expression of lineage-specific transcriptional programs or by de novo activation of cis-regulatory elements, favouring the establishment of an active (or repressive) chromatin state and ultimately changes in the gene expression pattern. In small-cell lung cancer (SCLC), copy number alteration-driven upregulation of the TF Nfib fosters the activation of evolutionary conserved cis-regulatory elements in primary tumour cells. These activated enhancers stimulate the expression of neuronal gene expression programs, which support metastasis burden [cis-regulatory elements.TFs play an important role in cancer progression, as they can alter the epigenetic landscape of single cells by modulating chromatin accessibility at s burden . Of impos burden  without high subpopulation within a tumour bulk was highlighted. In the mammary cell hierarchy, SOX10 plays an important role in the maintenance of multi-lineage potential during mammary gland development. During tumour progression, the SOX10high subpopulation of the tumour showed a higher level of dedifferentiation and features of epithelial-to-mesenchymal transition (EMT), linking subpopulation-specific TF activity to the aggressiveness of cancer [In another study, the overexpression of the oncogene c-MYC in immortalized human mammary epithelial cells resulted in an epigenetic reprogramming and a repression of lineage-specifying TFs. At the same time, MYC overexpression caused the reactivation of developmental pathways and the switch of the cancer cells towards a more aggressive stem-cell-like state ,58,59. If cancer . Besides the direct deregulation of TFs, multiple signals provided by the TME can support epigenetic reprogramming, fostering cancer cell plasticity and tumour progression. Among these, TGF-β signaling plays a central role in the induction of EMT, a reversible process important for cell migration . In healBesides the overexpression of oncogenes or the activation of TFs in response to deregulation of external stimuli, cancer treatments can also have a striking effect on the epigenetic landscape. In recent years, it became evident that drug-mediated epigenetic reprogramming imposes a major challenge for drug administration and cancer treatment, as long-term drug exposure can result in unwanted effects. Bi and colleagues showed that in luminal breast cancer the acquisition of endocrine resistance is linked to the reshaping of the enhancer activity, altering the transcriptional program . Mechanicis-regulatory elements controlling the expression of NOTCH target genes. Specifically, it has been shown that the KDM6 family H3K27 demethylases play a major role as drivers of enhancer reactivation [Comparable results were obtained by using vemurafenib, which triggered a stepwise reprogramming of a subpopulation of melanoma cells towards a resistant phenotype with an altered chromatin accessibility landscape identified by ATAC-seq, which led to dedifferentiation and subsequent activation of novel, until then silenced, pathways . Sharma tivation . Similartivation . Overallcis-regulatory elements to their target genes. At a higher level of chromatin organization, DNA is organized into topologically associated domains (TAD) in which multiple interactions are favoured, while limiting contacts with genomic regions positioned outside the same TAD [The eukaryotic genome is packaged into a highly organized hierarchical structure that is directly associated with the establishment and maintenance of regulatory networks and transcriptional regulation. Nucleosomes, a 147 bp DNA fragment wrapped around an octamer of histone proteins, are considered to be the most basic of the chromatin structure. At an intermediate level, the chromatin is organized in protein-mediated DNA loops that facilitate the interaction between genomic loci that lay distant in the linear genome, many of them linking same TAD . At a mesame TAD . The comsame TAD . Architectural proteins such as CTCF and SMC-family complex, cohesin, are known to play a critical role in the 3D organization of the genome, and several studies have demonstrated that the genetic or epigenetic alteration of CTCF binding can lead to disruption of 3D genome organization and may result in aberrant gene expression. Both CTCF and cohesion-binding sites suffer an exceptionally high mutational rate in cancer genomes ,77,78. CAlterations in local chromatin interactions are a hallmark of cancer genomes and have frequently been associated with activation of oncogenes and silencing of tumour suppressor genes. Recent studies have shown that structural variations (SVs) can modify the 3D chromatin structure by disrupting regulatory sequences that control higher-order chromatin organization . RecurreAs described above, epigenetic changes induced by extra- or intracellular factors can prime cells for the successful evasion from the primary site favouring the onset of disseminated tumour cells (DTCs) that can colonize distal sites to form metastatic lesions. After successful dissemination, DTCs gain cell plasticity, favouring their survival by an immune escape, to ultimately support metastasis overgrowth. For cancer cell homing, cells can reactivate development-associated transcriptional programs, which allow them to adapt to the new environment. This was shown for castration-resistant metastatic prostate cancer, in which enhancer hijacking and reactivation of developmental pathways resulted in successful homing of metastatic clones . In anotCancer samples consist of a complex and heterogeneous network of cellular interactions that govern tumour progression and metastasis formation. These complex ecosystems have traditionally been profiled using bulk experimentation studies that have only revealed an averaged cellular behaviour over thousands of cells, thus veiling the real intra-tumour heterogeneity and subsequently limiting our understanding of the biological implications and molecular mechanisms driving such functional cancer heterogeneity. The advent of single-cell transcriptomics and epigenomic methods has facilitated a comprehensive analysis of the various cellular programs and mechanisms of genetic regulation within heterogeneous cell populations at the single-cell level. In this section, we focus on recent evidence of the cell-to-cell genetic and non-genetic tumour heterogeneity and its implications on tumour growth, evolution, metastasis, drug resistance, and therapy, among others.Application of single-cell genome analyses in cancer research, which can determine whether mutations are in the same or different cells, confirmed that individual cancer samples are genetically heterogeneous, as previously inferred from bulk data . Single-The interplay between genetics, epigenetics, and environmental signals sets the gene expression pattern of a cell and thus determines the cell’s fitness and behaviour. Gene expression ITH typically reveals two main layers: very distinct clusters that correspond to different cell-types composing the cancer samples  and further subclusters that may represent the specific cellular states, elated to cell cycle, stress, hypoxia, epithelial differentiation, senescence, protein metabolism, and other dynamic programs . scRNA-sEpigenetic mechanisms allow for the regulation, maintenance, and inheritance of transcriptional programs. In contrast to genetic mutations, epigenetic changes are dynamic and potentially reversible, leading to heterogeneity during normal development, disease evolution, or in response to environmental stimuli. Loose epigenetic constraints in cancer cells are likely to enhance cellular plasticity and allow switching between cellular states, thus favouring cancer cell adaptability and resistance to therapy. Pioneering work by Corces et al.  demonstrTME can directly interact with tumour cells and influence malignant cell function and thus influence the behaviour of the tumour. Single-cell OMICs experiments have also revealed a certain degree of heterogeneity of the TME both within and between tumours . HeterogTumour eITH plays a key function in tumour progression and metastasis.Genomic heterogeneity plays an important role at the onset of tumorigenesis, which is later driven by non-genetic changes.Proper dissection of the tumour at the epigenetic level allows for personalized therapeutical approaches and prediction of tumour burden.The development of single-cell approaches facilitates the identification of tumour subpopulations.Single-cell OMICs has revealed a far larger clonal complexity of the tumours than previously expected.Our increasing understanding of the subclonal dynamics of cancer and its diverse trajectories using single-cell omics has enabled a better characterization of mechanisms of drug resistance and metastasis that will offer innovative therapeutic opportunities.Epigenetic alterations can further diversify the genetic subclonal structure of the tumours and generate phenotypically distinct subclones with differing therapeutic resistance, thus underscoring the importance of targeted epigenetic drugs for future combination therapies.Analysis of the TME at the single-cell resolution indicates that the composition of the TME, as well as its interplay with the tumour cells, can also modulate the therapeutic response.Imaging approaches allow for the spatial resolution and the analysis of the interplay between tumour and TME.Key points: Identification of the main epigenetic mechanisms driving resistance, relapse and metastasis burden.Linking phenotypic features and molecular characterization of the resistant and metastatic cells.Spatially and temporally resolved single-cell studies to better understand the role of TME in tumour evolution and leveraging this information for better combination therapies.Understanding the relationship between genomic and epigenomic alterations in cancer cells.The translation of single-cell omics into the clinical routine is still hampered by their limited high-throughput, relatively high costs, and lack of technical and computational standarized procedures.Challenges:Characterization of tumour subpopulations and their epigenetic state is still a major challenge of today’s research and therapy development. Evidence suggests that tumours and the surrounding microenvironment comprise a heterogeneous mixture of different subpopulations, as shown by single immunofluorescence imaging and more recently by sc-OMICs approaches. Recent studies have shown that changes in the genome, epigenome, and transcriptome of rare subpopulations, all of which were masked and undetectable by bulk genomic analysis beforehand, can drive cancer growth, progression, and drug resistance and thus have a profound implication in cancer biology. Specifically, cancer epigenomic studies at single resolution have come to demonstrate that epigenetic heterogeneity within individual cancer samples is functional, further increases the plasticity of transcriptional programs, and thus influences drug sensitivity and clonal dynamics of cancer evolution. Moreover, recent developments in spatially resolved single-cell genomics promise to help us to unravel the spatio-temporal distribution of epigenetic and phenotypic features in single-cells and will thus improve our understanding of the association between cellular composition and spatial distribution of the tumour and surrounding microenvironment cells. This knowledge can be translated into the clinic by providing customized treatment options for cancer patients, targeting the different tumour subpopulations identified.


In-hospital cardiac arrest in patients with COVID-19 presents significant challenges to health care teams. Airborne precautions can delay patient care, place providers at high risk of virus exposure, and exacerbate an already stressful environment. Within the constraints of an ongoing pandemic, an efficient educational program is required to prepare health care teams for airborne isolation code blue.This simulation was conducted in a room on the target unit using a CPR manikin to represent the patient. A “talk-through walk-through” scripted simulation directed learners  through a resuscitation using an airborne isolation code blue protocol. Key scripted events prompted role identification, communication, and item transfer. Learners self-assessed their airborne isolation code blue knowledge and skills and their confidence in providing quality care while maintaining safety using a pre-/posttraining 5-point Likert-scale survey.p < .001) increase in percentage selecting agree/strongly agree for all statements related to knowledge and skills specific to airborne isolation code blue protocol, as well as confidence in providing care while keeping themselves and their colleagues safe.We trained 100 participants over a 5-month period, with 65 participants surveyed . Following training, participants had a statistically significant (Our simulation program allowed a small number of educators to feasibly train a large number of learners, let learners practice required skills, and improved learners’ self-assessed knowledge, skills, and confidence regarding quality and safety of care. Despite the arrival of successful vaccines, new variants are spreading worldwide, and there are reports of serious illness even among vaccinated individuals.11 The American Heart Association (AHA) suggests a two-team model with fewer personnel inside the code room to decrease direct exposure and ration the use of PPE.12 Without adequate education, however, such a protocol may present more risk than benefit.7 Our hospital initially distributed our AHA-based COVID-19 code blue protocol through several modalities, including training at nursing huddles, flyers, and a training video  to refine training. Initially, we found that learners struggled to focus on the key isolation-specific variables defined as our critical actions see . We thenMaterials included a CPR manikin to perform chest compressions, a pair of walkie-talkies, a defibrillator, a backboard, simulated medications (epinephrine and amiodarone), and lab tubes . The uniSessions were led outside the room by a single instructor, an internal medicine hospitalist or pulmonary critical care attending familiar with the airborne isolation code blue protocol. Inside the room, a standardized participant who acted as an ICU nurse provided prompts as needed to facilitate performance of the critical actions. The standardized participant was usually another program committee member, but when they were not available, this role was filled by an internal medicine resident (on three occasions) who was not a member of the code team. They received presession training to review the isolation code blue protocol and ICU nurse role in the simulation script, including prompts.From August to December 2020, we held 12 training sessions, each designed to accommodate two to four internal medicine residents and four to eight acute care, ICU, and STAT nurses. All learners were already in the hospital for clinical duty; therefore, sessions were limited to 30 minutes, with an option to stay later for additional questions or practice. Residents were preassigned to participate in groups of two within their first week on ward rotations during which they would serve as code blue leaders. Nurses were assigned by unit nurse managers to maximize the number of participants over time. Additional attendees, including respiratory therapists and advanced practice providers, were welcome to attend . SessionAt the start of each session, learners were given a paper handout with the airborne isolation code blue protocol diagram  and codeA role-specific critical action checklist  was deveAn anonymous postsession survey was shared with participants via a QR code link or paper . ParticiThe debrief was led by the instructor, with input from the standardized participant ICU nurse. Participants reviewed the unique differences between airborne isolation and routine code blue events. They were asked to describe what made communication and item transfer in a two-team environment successful, what challenges arose in these areas or with the use of PPE, and how those challenges could be addressed. Learners were encouraged to raise questions or concerns and share their real-life experiences to inform protocol implementation or adjustments. The debrief was designed to be a minimum of 5 minutes to facilitate return to clinical duties; however, learners were given the option to remain longer and usually stayed for approximately 15 minutes.One hundred unique learners were trained during 12 training sessions from August to December 2020, including the 29 internal medicine residents who rotated on the acute care service at our hospital during this period. All learners successfully completed training and demonstrated the critical actions, as judged by the instructor in real time. Many learners initially struggled to complete the critical actions, particularly using the walkie-talkies to communicate. Learners were prompted to continue the simulation until all groups were able to complete all critical actions, as assessed by the instructor. During the nine sessions within the survey period of October-December 2020, 43 of 65 participants (66%) responded see . Of thesn = 2, 12%), where to find PPE , ability to communicate effectively , and ability to transfer items into and out of the room  in the percentage of participants selecting agree or strongly agree for all statements. Improvement was independent of learner type (residents and nurses). There was no difference in posttraining scores between residents and nurses, except knowledge of where to acquire PPE .Participants’ self-assessment of pretraining protocol knowledge and skills is depicted in Participants’ confidence in their ability to provide high-quality care while maintaining safety for themselves and colleagues similarly improved after training. More than 95% of participants with prior airborne isolation code blue experience selected agree or strongly agree for all posttraining statements . The sur2This talk-through walk-through scripted simulation training program allowed for efficient education and practice of airborne isolation code blue protocol for a large portion of resident and nursing staff in an urgent time frame. It was feasible to implement despite barriers such as physical distancing, staffing shortages, and financial strain that would limit high-fidelity simulation lab training. The program was designed and implemented by two physicians and one nurse, who spent no more than 1 hour per week conducting sessions. The program did not require the purchase of new equipment or technician support. Participants spent just 30 minutes away from work, requiring no special coverage. In the midst of an unprecedented global crisis, this simple design can serve as a framework to rapidly implement COVID-19 code blue training, particularly in low-resource communities at highest risk for outbreaks.Following simulation training, all participants reported increased self-assessed knowledge of and preparation for conducting an airborne isolation code, as well as increased confidence in quality of care and provider safety. It is notable that less than a quarter of residents reported an adequate baseline knowledge of essential team leader elements such as who should enter the room and how to communicate between inside and outside teams. Residents also rated a particularly low baseline knowledge of where to acquire appropriate PPE. This was notable because during the 2020–2021 winter surge, supply shortages limited the quantities of PPE available directly outside of patient rooms. Ensuring that residents knew where to locate additional emergency PPE or the unit PPE bucket was essential to facilitating their efficient and safe entry into the code room. These low baseline scores were likely related to frequent rotation of residents among hospitals and services, limiting reinforcement of airborne isolation practices. After training, residents showed robust improvement in these areas. A simulation script allowed us to control for the large cognitive load of a traditional mock code, focusing learners on the critical actions related to airborne isolation code blue protocol. Free-text survey feedback supported this benefit of a scripted structure.Our unique simulation design let us hold in situ sessions despite the significant resources and time typically required for mock codes. This allowed learners to locate the unit's equipment and PPE and to navigate the specific unit doors to safely enter, transfer items, and communicate during the code. When nonclinical environments were used, maintaining door type and function allowed us to proceed with these same benefits despite a surge in hospital census. In addition to being cost effective and resource efficient, our unique simulation format allowed us to stress-test our system and training program in a compressed time frame. Based on observations during early sessions, we refined our protocol and tailored our program to include just-in-time walkie-talkie training for residents. The talk-through element also allowed us to compensate for challenges to preeducation: Despite encouragement, almost half of our learners had not had any prior education. This likely reflects real-world conditions of heavy clinical workload during the COVID-19 crisis. While learners with pretraining had higher posttraining self-assessment and confidence, learners with no prior experience still showed significant improvement with our simulation format by obtaining preeducation  and simulation within a single session.27 and they add to a growing body of evidence supporting the utility of simulation programs to prepare for COVID-19 cardiac arrest and other rare resuscitation events.28 Unlike previous work, however, our talk-through walk-through structure and scripted simulation is far simpler to implement in time-, personnel-, and resource-constrained settings such as those with current outbreaks due to low vaccination rates. While we cannot directly compare our outcomes to a traditional high-fidelity simulation structure, some of our highest-risk communities are less likely to have a dedicated simulation lab. Nursing and physician leadership at other hospitals can easily adopt our protocol and materials or customize the simulation script to reflect their own airborne protocol. Additionally, this approach is easily adapted to other airborne isolation code events, such as patients with M. tuberculosis or disseminated varicella infections, and can be used to rapidly implement other changes to emergency response.Our learner-reported improvements are similar to those seen in prior cardiac arrest simulation literature,Our work has several limitations. This was a single-center program, and although it is easily adaptable to other environments, its results may not be generalizable. Our simulation design was heavily dictated by the resource, time, and personnel demands of an ongoing pandemic. Similarly, these barriers prevented the use of pre- and posttraining objective assessments to quantitatively document improvement in performance of large numbers of learners in diverse roles, due to clinical need and overall program goals. Therefore, we utilized self-assessment in a retrospective survey tool, which has inherent limitations of bias. While this was an appropriate approach to address our secondary objectives of improved learner confidence in ability to provide quality care and maintain provider safety, future work during less strained situations would benefit from more rigorous skill-assessment methods to optimize the program. As resources become more accessible, future work could also include incorporating formal presession education.Overall, our simulation program was successful at efficiently educating resident and nursing staff regarding airborne isolation cardiac arrest using minimal resources and personnel. It increased the number of learners exposed to airborne isolation code blue protocol and improved self-assessed knowledge, skills, and confidence to participate in real-life airborne isolation cardiac arrest.Protocol Diagram.docxTraining Video.mp4Simulation Case Template.docxSimulation Images.pdfAction Priorities.docxSimulation Script.docxSurvey.docxAll appendices are peer reviewed as integral parts of the Original Publication.


The composition and organisation of the extracellular matrix (ECM), particularly the pericellular matrix (PCM), in articular cartilage is critical to its biomechanical functionality; the presence of proteoglycans such as aggrecan, entrapped within a type II collagen fibrillar network, confers mechanical resilience underweight-bearing. Furthermore, components of the PCM including type VI collagen, perlecan, small leucine-rich proteoglycans—decorin and biglycan—and fibronectin facilitate the transduction of both biomechanical and biochemical signals to the residing chondrocytes, thereby regulating the process of mechanotransduction in cartilage. In this review, we summarise the literature reporting on the bidirectional reciprocity of the ECM in chondrocyte mechano-signalling and articular cartilage homeostasis. Specifically, we discuss studies that have characterised the response of articular cartilage to mechanical perturbations in the local tissue environment and how the magnitude or type of loading applied elicits cellular behaviours to effect change. In vivo, including transgenic approaches, and in vitro studies have illustrated how physiological loading maintains a homeostatic balance of anabolic and catabolic activities, involving the direct engagement of many PCM molecules in orchestrating this slow but consistent turnover of the cartilage matrix. Furthermore, we document studies characterising how abnormal, non-physiological loading including excessive loading or joint trauma negatively impacts matrix molecule biosynthesis and/or organisation, affecting PCM mechanical properties and reducing the tissue’s ability to withstand load. We present compelling evidence showing that reciprocal engagement of the cells with this altered ECM environment can thus impact tissue homeostasis and, if sustained, can result in cartilage degradation and onset of osteoarthritis pathology. Enhanced dysregulation of PCM/ECM turnover is partially driven by mechanically mediated proteolytic degradation of cartilage ECM components. This generates bioactive breakdown fragments such as fibronectin, biglycan and lumican fragments, which can subsequently activate or inhibit additional signalling pathways including those involved in inflammation. Finally, we discuss how bidirectionality within the ECM is critically important in enabling the chondrocytes to synthesise and release PCM/ECM molecules, growth factors, pro-inflammatory cytokines and proteolytic enzymes, under a specified load, to influence PCM/ECM composition and mechanical properties in cartilage health and disease. Articular cartilage, located at the ends of diarthrodial joints, has a unique biochemical composition that confers its functional properties, the principal function being the dissipation of mechanical forces evenly across the joint surface. The composition of the extracellular matrix (ECM) in articular cartilage is critical to its function, and it is well known that the presence of proteoglycans embedded within a collagen fibrillar network provides biomechanical resilience. Furthermore, the spatial distribution of these ECM molecules within the tissue also facilitates its capability to withstand mechanical loads, as well as perceive biomechanical signals transducing them to the chondrocytes (only cell type in cartilage) to effect cellular responses. This bidirectionality within the ECM is critically important in enabling the chondrocytes to sense load application, including altered loading patterns, but also to influence ECM composition as a consequence of mechanical cues .Prg4) gene, colocalises with cartilage oligomeric protein (COMP) and binds FN and type II collagen . F. F167]. This dramatic reduction in cartilage ECM content following mechanically mediated joint injury has been correlated with significant effects in the PCM. Using the destabilisation of the medial meniscus (DMM) murine PTOA model, Chery et al. (2020) observed a decrease in the mechanical properties of the PCM, although PCM composition was maintained; type VI collagen, perlecan and BGN remained localised to the PCM at 8 weeks, concomitant with reduced aggrecan content . A signi−/−Dcn) and inducible DCN knockout (iKODcn) mice subjected to medial meniscus destabilisation i signalling in response to osmotic stress, which was more pronounced with age (>2 weeks old) [2+]i signalling in wild-type mice which was further compounded in the −/−Dcn mice [2+]i signalling responses. Within the ECM, aggrecan and its chondroitin sulphate sGAG moieties provide the net negative charge facilitating sequestration of cations such as Ca2+ for the subsequent activation of ion channels and transmission of mechanical signals. Without these biophysical properties, cartilage chondrocyte mechanotransduction would be severely compromised.With OA, structural and biochemical profiles of the PCM are altered, and degeneration of the PCM is reportedly one of the first events to occur resulting in aberrant chondrocyte mechanotransduction and ultimately cartilage degradation ,238,239.eks old) . Interes−/− mice ; this deArticular cartilage responds to mechanical perturbations in the local tissue environment, sensing alterations in the magnitude or type of loading applied, and eliciting cellular behaviours to effect change. In situ, the chondrocytes reside within a dynamic mechanical environment comprising a combination of compression, hydrostatic pressure, shear stress, osmotic stress and tensile strain. Numerous studies have demonstrated the pivotal nature of cell–matrix interactions and the distinct properties conferred by the PCM in regulating the chondrocytes’ mechanical environment, influencing cartilage homeostasis and joint health. Specific PCM components, e.g., type VI collagen, perlecan and SLRPs, are instrumental in facilitating the sensing of mechanical signals and orchestrating appropriate mechano-signalling responses. Insight into composition and structure of the PCM, in conjunction with its mechanical properties in cartilage health and pathology, has been instrumental in our understanding of the reciprocity of the PCM/ECM and chondrocyte mechanotransduction. Furthermore, such studies have revealed the complex interplay of loading, inflammation and growth factors on the biochemical and biomechanical hierarchy of articular cartilage. This knowledge can be effectively utilised to guide development of small molecule therapeutics to prevent early remodelling of the PCM, induced by injurious loading, to delay or halt PTOA progression. Furthermore, understanding how physiological loading promotes PCM/ECM synthesis resulting in deposition of a highly organised matrix will aid development of cell constructs containing the hierarchical structure of cartilage for use in tissue-engineering strategies. Although we do not have a complete understanding of chondrocyte behaviour in response to physiological and non-physiological loading, this review demonstrates a clear bidirectional reciprocity between the chondrocyte and the PCM/ECM in regulating structural, biomechanical and functional characteristics of articular cartilage and how this can impact tissue health and pathology.


Abiotic stresses, including drought, extreme temperatures, salinity, and waterlogging, are the major constraints in crop production. These abiotic stresses are likely to be amplified by climate change with varying temporal and spatial dimensions across the globe. The knowledge about the effects of abiotic stressors on major cereal and legume crops is essential for effective management in unfavorable agro-ecologies. These crops are critical components of cropping systems and the daily diets of millions across the globe. Major cereals like rice, wheat, and maize are highly vulnerable to abiotic stresses, while many grain legumes are grown in abiotic stress-prone areas. Despite extensive investigations, abiotic stress tolerance in crop plants is not fully understood. Current insights into the abiotic stress responses of plants have shown the potential to improve crop tolerance to abiotic stresses. Studies aimed at stress tolerance mechanisms have resulted in the elucidation of traits associated with tolerance in plants, in addition to the molecular control of stress-responsive genes. Some of these studies have paved the way for new opportunities to address the molecular basis of stress responses in plants and identify novel traits and associated genes for the genetic improvement of crop plants. The present review examines the responses of crops under abiotic stresses in terms of changes in morphology, physiology, and biochemistry, focusing on major cereals and legume crops. It also explores emerging opportunities to accelerate our efforts to identify desired traits and genes associated with stress tolerance. Arabidopsis, and this basic knowledge needs to be accelerated to make the crop plants resilient to abiotic stresses in target growth environments. There is a need to explore those aspects of stress adaptation that have not been effectively utilized or employed to improve key food grain crops.Major abiotic stresses that are likely to be amplified by climate change and can destabilize crop yields include drought, extreme temperatures, flooding, waterlogging, soil salinity, acidity, mineral toxicity, and nutrient deficiency. Global climate change and environmental degradation are intensifying the severity of abiotic stresses that adversely affect the growth, development, and productivity of crop plants. The increasing frequency of extreme weather events due to climate change is expected to cause severe risks to sustainable crop production for major cereal and leguminous crop species. Losses in production caused by abiotic stresses may exceed 40% , and henMicrobes that are associated with plants in natural habitats under diverse environments exhibit tremendous capabilities to cope with stresses caused by environmental factors. Since plants must interact with microbes, microbes are believed to modulate plant defense mechanisms to protect against adverse external conditions ,11. WithPlants experience abiotic stresses when they are exposed to supra- or sub-optimal levels of environmental factors such as temperature, soil moisture, and salts in the soil ,13. It iDroughts are a major challenge faced by most food crops that are sensitive to a soil moisture deficit. The impact of droughts on the final yield and various physiological and biochemical processes of crops depends on its intensity, timing, and duration . HoweverProjected increases in higher ambient temperatures worldwide are likely to drastically reduce crop productivity ,23,57. T2+, Mg2+, Na+, and S remains unaffected [High temperatures affect photosynthesis and its components in rice , wheat , maize , beans , and theaffected .Salinity is a significant abiotic stress that restricts crop growth and productivity and is characterized by an excessive concentration of soluble salts in the soil that suppresses plant growth in many irrigated, arid, and semi-arid regions of the world . The ext−1 led to a decrease in rice grain yield [+ and Cl−. High Na+ concentrations prevent the absorption of K+ ions, which are highly essential for growth and development [An increase in the EC above 0.88 dS min yield . The grain yield . Salinitin yield , wheat [in yield , maize [in yield , the fabin yield , the chiin yield , and thein yield ,126. Thielopment .+/K+, Na+/Mg2+, Na+/Ca2+, Cl/H2PO4, and Cl−/NO−3, which adversely affects plant cellular processes [+ from the leaf and to discriminate between K+ and Na+. It is well-known that salinity affects plant growth through low soil solution osmotic abilities and nutritional imbalances [The interference of salts with the nutritional homeostasis of plants increases ionic ratios such as Narocesses ,129. Crorocesses . These dbalances . In cerebalances , wheat [balances , maize [balances , and thebalances .Waterlogging and submergence causes substantial yield losses in food grain crops. Climate change scenarios predict increases in future incidences and intensities of floods, especially in the tropics and subtropics . Most dr2. Therefore, flood-damaged plants have a lower rate of aerobic cellular respiration than normal plants. Low CO2 concentrations in flooded leaves subsequently limit photosynthesis. Flooding leads to an energy crisis within the cells of plants [Underwater plant cells that carry out photosynthesis do not readily exchange oxygen and COf plants . Waterlo2 in plant cells and induces the accumulation of oxygen free radicals. Plants under waterlogged conditions experience increased cellular damage from reactive oxygen species [The waterlogging reduces photosynthesis due to stomatal closure, as well as abscisic acid (ABA), ethylene, and active oxygen species production. In addition, stomata closure often restricts CO species .Plants are constantly exposed to various environmental stresses such as salinity, drought, cold, and high temperatures. The impact of abiotic stresses on the grain yield of cereals and legumes is shown in 2 levels, which are advantageous when combined with other stresses such as salt or high light [In comparison, relative to each of the stresses applied separately, certain stress combinations may benefit plants. Examples include elevated COgh light . Salinitgh light .Plants are constantly under pressure from environmental stresses, and they tolerate or resist stress by various adaption and acclimation mechanisms. Plants have evolved complex physio-biochemical and molecular strategies to neutralize the effects of abiotic stress ,171. ThePlants adapt to droughts by adjusting their phenology, morphology, and physiology at the cellular and molecular levels. The drought-induced inhibition of growth as well as yield reductions can be attributed to adverse effects on plant functions and processes, particularly plant water uptake, water use efficiency, and the partitioning of biomass to grains.Plants, including cereals and legumes, tend to escape droughts by curtailing the crop growth duration or accelerating phenological phases, referred to as a flexibility in phenology . This feThe plant adjusts to droughts by lowering water loss and getting more water through root uptake. Adaptive traits are used to set the background of a “low transpiration rate in water-saving plants” and an “osmatic adjustment in water-spender plants” to prevent dehydration ,174.Plants mostly rely on leaf relative water content, osmotic adjustments, and root architecture to enhance the yield under drought stress . ReducinDrought-tolerant plants exhibit adaptive root properties, including long roots, high densities of roots, and intense rooting systems ,181,182.The cellular dehydration of tissues occurs when plants are exposed to extreme temperatures, droughts, and salinity. The plant cell produces osmolytes such as sugars, proteins, nucleic acids, and amino acids to protect from dehydration, as reported in wheat . An osmo2O2 metabolism [Reactive oxygen species (ROS) such as singlet oxygen, hydrogen peroxide, superoxide, and hydroxyl radicals are involved in various cellular functions ,197. ROStabolism . Rezayiatabolism  reportedtabolism  reportedtabolism ,203, seltabolism ,204 and tabolism  also pla2O2 [The plant possesses antioxidant machinery with the enzymatic and non-enzymatic components to mediate redox signaling and ROS homeostasis linked to acclimation responses to abiotic stressors. In response to stress, several antioxidative enzymes are produced by the plant. Superoxide dismutase (SOD), catalase (CAT), and peroxidases (POX) are among the enzymatic components of antioxidant systems that regulate the homeostasis of ROS within organisms, as reported in wheat ,205, ric2O2 ,208,209.2O2 .2O2) generation are typical heat stress incidents [At the cellular level, singlet oxygen, superoxide, hydroxyl ion, and hydrogen peroxide  is the key hormone considered to regulate the response of plants to abiotic stresses. Increased levels of endogenous ABA under drought stress conditions have been reported in many plant species, which include grain crops like sorghum , rice 2, barley The exogenous application of ABA may also increase responses to droughts in wheat ,228. ABAIPT) delays cell senescence caused by droughts and improves crop yields [Cytokinin (CK) postpones the premature senescence of leaves and death during drought stress and promotes adaptive traits that help enhance grain yields. The increase in endogenous levels of CK through the expression of the CK biosynthesis gene isopentenyl transferase , or stress-induced proteins. Plants under stress tend to produce less normal proteins and up-regulate genes associated with HSPs . About 2Exogenous applications of osmoprotectants, phytohormones, signalling molecules, and trace elements have shown positive impacts on plants grown under heat stress, as they have growth-promoting and antioxidant abilities. The heat tolerance in plants may be increased by the exogenous application of osmoprotectants . Several2O2) generation and reactions are typical heat stress incidents [At the cellular level, singlet oxygen, superoxide, OH, and hydrogen peroxide  in the root cortex, expanding the stem (hypertrophy), forming adventitious roots near the soil surface, and root tip death . Rice haIn plants, abiotic stress tolerance is a complex trait involving many different metabolic pathways and cellular and molecular components. Abiotic stresses commonly induce various responses at the morphological, physiological, biochemical, and genomic levels. For several decades, the plant research community has amassed a highly comprehensive understanding of the physiological and biochemical mechanisms that facilitate productivity maintenance in response to several abiotic stresses like droughts, flooding, heat stress, cold, salinity, and heavy metals. Understanding the abiotic stress tolerance mechanisms laid the foundation for the development of climate-resilient crop varieties . The conVrn), photoperiod (Ppd), and earliness per se (Eps), and the genetics of these traits have been studied extensively, particularly in cereals [Early flowering or maturity (EF/EM) is the most critical phenological trait/mechanism exploited by breeders for the development of short-duration varieties which can escape abiotic stresses, particularly droughts and heat stress. Early flowering and seed set before an upcoming drought event are important in legumes  and cere cereals ,333,334. cereals  gave an  cereals ,335,336. cereals . Four cuBW507 and BW508, in barley which were reported to carry two independent early-flowering mutant loci, mat-b.7, and mat-c.19, respectively. They mapped the mutation in BW507 to a 31 Mbp interval on chromosome 2HL and concluded that BW507 has a deletion of Mat-c, which is an ortholog of Antirrhinum majus CENTRORADIALIS (AmCEN) and Arabidopsis thaliana TERMINAL FLOWER1 (AtTFL1) and is a key gene in regulating early flowering. There is evidence that evolution can favor EF/EM traits in native populations of plants even without the pressure of oncoming drought stress [Vigna taxa more easily acquired phenological traits for a short life cycle to escape droughts and heat stresses compared with acquiring physiological traits.Matyszczak et al.  identifit stress . Zonnevet stress  concludeVariations in root system architecture can be explored for the development of climate-resilient and abiotic stress-tolerant crops by improving water and nutrient use efficiency . The priPhule et al.  observedPolania et al.  identifiPhotosynthesis in higher plants is sensitive to various abiotic stresses. The photosystem II (PSII) is the most sensitive to desiccation. Its sensitivity could be strongly associated with the desiccation severity . ChloropDro1) gene [Dro1 homologs in the improvement of salt tolerance in rice has been demonstrated [In recent years, the use of root system architecture for the improvement of stress tolerance in crop cultivars has gained attention. The discovery of the deep root 1 (o1) gene  has beeno1) gene . Similaro1) gene . The utinstrated .SUB1) locus in rice, conferring tolerance to complete inundation (submergence) [SUB1A was identified and cloned from a submergence-tolerant cultivar, i.e., FR13A, and it had been developed through selection from farmers’ variety “Dhallaputia” grown in Odisha (India). SUB1A is the dominant gene(s), and fine mapping of this gene on chromosome 9 has been completed. A marker-assisted backcross breeding approach is now being successfully exploited for the development of high-yielding submergence-tolerant rice cultivars. Under mild stress (5–6 days submergence), plant mortality in rice is generally very low, yet extensive leaf damage occurs. However, the damage is almost nil in cultivars with SUB1 QTL genes due to the maintenance of higher activities of antioxidant enzymes. Prolonged water stagnation decreases the grain yield in cultivars with SUB1 (Swarna-Sub1) compared to those without SUB1 . Sarkar et al. [Sub1 is suitable for flash flood conditions. They also concluded that the maintenance of chloroplast integrity could be a better option for predicting the plant survival under submergence. Some researchers believe that the ideal combination for an adaptation to complete flooding is submergence tolerance  together with some elongating abilities [The physiological and molecular responses of rice to flooding have been extensively studied , providiergence) ,352. SUBr et al.  suggestebilities .SUB1A-1 suggests that SUB1A-1 from wild species might have introgressed around the Ganges Basin and subsequently spread to other areas of South Asia [SUB1A-1 is absent in submergence-tolerant accessions of wild rice with the CC genome (O. rhizomatic and O. eichingeri) and the CCDD genome (O. grandiglumis) [SUB1A-independent mechanism in these rice species. Future elucidation of the SUB1A-independent mechanism may contribute to the future breeding of cultivated rice with strong flash flood tolerance.The geographical distribution of these accessions harboring uth Asia . By contiglumis) ,356, sugLGF1), a wax synthesis gene involved in the leaf hydrophobicity and formation of the gas films necessary for gas exchange and underwater photosynthesis, is another important step towards developing flood-tolerant varieties [LGF1 briefly gave insights into the potential shoot and root traits that can improve submergence tolerance in rice. Kuroha and Ashikari [A recent significant discovery of the cloning of the Leaf Gas Film 1  and type G proteins (GTGs)  were conAn orthogonal receptor is an engineered receptor that can bind specifically to a synthetic ligand, which cannot interact with the natural receptor. The orthogonal receptor is not activated by the basal level of the endogenous ligand. The orthogonal receptor is helpful since they get activated by synthetic ligands at low concentrations, while the natural receptors require relatively higher doses of synthetic ligands for activation. The orthogonal receptor can be used to design crops for resilience to multiple abiotic stresses, where synthetic agonists or antagonists can be used to induce a specific physiological process such as metabolite production and tolerance to abiotic stress . These sPlants have evolved sophisticated stress response strategies and harbor genes that encode transcription factors (TFs) to regulate the expression of stress-responsive genes. TFs could be candidates for enhancing resilience to multiple abiotic stresses. Recently, the TF modulation and overexpression approaches have been employed in crop plants; however, the diversity of TFs largely remains unexplored. TF families such as NAC, MYB, WRKY, bZIP, and ERF/DREB have been successfully characterized for their roles in eliciting abiotic stress responses. About 10% of genes encode TFs, which play an important role during different stages of the plant life cycle for a specific function . TherefoAdvanced technologies like chromatin immunoprecipitation with massively parallel sequencing (CHIP-Seq) and next-generation sequencing (NGS) could be beneficial for genomic region identification for deciphering the role of TFs. The CRISPR/Cas  gene-editing tool can be used to modulate TFs. In addition, the functional redundancy of TFs needs to be addressed. Although previous analyses of TF overexpression in response to a specific stress have been very informative, studies are required to investigate whether the overexpression of stress-related TFs in transgenic plants enhances stress tolerance and growth without any yield penalty. Further studies are required to understand the mechanisms of the action of several TFs and their role in enhancing stress tolerance in food grain crops.The molecular breeding and molecular genetics for the genetic enhancement of plants for yield stability under adverse environmental conditions could potentially capture the effective resilience to abiotic stress. Plants generally reduce vegetative growth and accelerate reproductive development under stress. The genetic variations and underlying mechanisms that enable drought-resilient plants to conserve soil moisture and delay the accumulation of biomass until grain filling largely remains unclear. Higher yields under well-watered conditions and under a moderate drought at the time of flowering were achieved in corn that expresses a metabolic enzyme that converts trehalose-6-phosphate (T6P) to trehalose in the phloem companion cells at the base of the ear and in developing florets . The modRecent advances in molecular genetics and genomics have enabled the identification of a complex signaling network associated with plant growth and development. Many genes have been identified and characterized for their role in abiotic stress tolerance, employing genomics and molecular genetics. However, efforts should be made to unravel the crosstalk between the transcriptional circuitries for biomass production and abiotic stress responses. This knowledge could serve as a valuable resource to eventually custom design the crop plants for higher biomass production with less water use in a more sustainable manner under adverse environmental conditions.The adaptation of plants to various abiotic stresses is a coordinated response involving many genes and their interactions with various environmental factors during the entire life span of crop plants ,370. AccPlant cell wall polymers form a significant component of plant biomass. The composition and amount of these polymers in the cell wall change with the developmental stage of plants and in response to stress conditions . There iThe challenge of improving abiotic stress tolerance in crops must be addressed with an understanding of the underlying complexities and with care taken to avoid grain yield penalties resulting from the introgression of relevant traits. A feasible approach to addressing this challenge should include the grain yield and the traits specific to targeted agro-ecologies. Although the possibility of improving stress tolerance has been successfully demonstrated through transgenic approaches, breeding varieties tolerant to abiotic stress is difficult. This has been attributed to the underlying complex mechanisms that influence the relevant traits and associated genes, which often act in coordination. There is ample scope for integrating different omics strategies to increase plant stress tolerance substantially, as marker-assisted selections by employing stress-related genes and QTLs are becoming more routine activities in the crop breeding program. Advanced tools such as CRISPR/Cas techniques for the modification of genes are becoming more relevant for the genetic improvement of abiotic stress tolerance in plants.Recent advances in omics approaches, including genomics, proteomics, and phenomics have provided new opportunities for understanding abiotic stress responses in plants at a different scale, from the cell level to the whole plant level. Designs for stress-tolerant crops can be substantially improved with the additional insights into the mechanisms of stress tolerance in plants obtained through deep sequencing technologies and other omics approaches, such as metabolomics . InformaThe existing knowledge gaps that are hindering the application of omics for abiotic stress tolerance in crop plants must be bridged through a system biology approach. Tools and methods for high throughput phenotyping must be optimized for the identification of relevant genes and their utilization in breeding different crops. This is likely to be aided by the emerging tools for extensive data analyses, which employs machine learning algorithms. Conventional approaches to crop production are now being complemented by remote sensing tools that are integrated for crop stress monitoring. Advances in imaging and sensor technologies, high throughput phenotyping, and remote sensing tools can enable rapid field-scale estimations of plant health and plant stress susceptibility or tolerance, guiding crop management decisions under an abiotic stress environment. The systematic evaluation of extensive germplasm collections to identify tolerant genes and genotypes, and the development of targeted breeding programs to enhance abiotic stress tolerance using traditional and novel methods, are both essential for increasing the yield and stress tolerance of food grain crops.