Engineering Stress Tolerance Research Articles

Differential Physiological and Molecular Processes in the Root May Underlie Contrasting Salt Tolerance in Two Egyptian Rice Cultivars at the Seedling Stage

This study aimed to compare various responses of two Egyptian rice accessions bred for high yields, Sakha108 and Giza177, to salt stress at the seedling stage. Twenty-eight-day-old seedlings of two cultivars were grown in a hydroponic medium under control conditions (no NaCl) and salt stress (75 mM NaCl) for 12 days. Growth (dry weight), Na+ and K+ concentrations, enzymatic and non-enzymatic antioxidants, malondialdehyde (MDA), hydrogen peroxide (H2O2), and expression of Na+ and K+ transport-coding genes were recorded. Sakha108’s growth (18% rise from control) was significantly higher than Giza177’s. Both cultivars accumulated similar amounts of Na+ in the leaves and sheaths, however, Sakha108 had higher Na+ concentrations in the roots than Giza177 (13.3% higher). Root K+ concentration dropped dramatically (~ 2-fold reduction) in Giza177 roots while remaining unchanged in Sakha108 roots. The concentrations of H2O2 (root) and MDA (leaf and root) were higher in Giza177 than in Sakha108, although the difference was not statistically significant. Proline and total flavonoid (TF) contents in Sakha108 roots were greater than those in Giza177 roots. The expression of OsHKT1;5 and OsHKT2;1 genes declined in both cultivars, whereas expressions of OsSOS1, OsNHX1, and OsHAK7 were induced in Sakha108 but, except for OsHAK7, were repressed in Giza177. Combined, these findings suggest that Sakha108 is more resistant to salt stress than Giza177 is, and that this variation in tolerance may have its origins in the root systems of the two cultivars. Thus, these adaptive traits in the root of Sakha 108 could be explored for engineering stress tolerance in susceptible but high-yielding rice cultivars.

M6A RNA methylation counteracts dark-induced leaf senescence in Arabidopsis.

Senescence is an important physiological process which directly affects many agronomic traits in plants. Senescence induces chlorophyll degradation, phytohormone changes, cellular structure damage, and altered gene regulation. Although these physiological outputs are well defined, the molecular mechanisms employed are not known. Using dark-induced leaf senescence (DILS) as the experimental system, we investigated the role of N6-methyladenosine (m6A) mRNA methylation during senescence in Arabidopsis (Arabidopsis thaliana). Plants compromised in m6A machinery components like METHYLTRANSFERASE A (mta mutant) and VIRILIZER1 (vir-1 mutant) showed an enhanced DILS phenotype. This was accompanied by compromised chloroplast and photosynthesis performance in mta as well as accumulation of senescence-promoting camalexin and phytohormone jasmonic acid after dark treatment. m6A levels increased during DILS and destabilized senescence-related transcripts thereby preventing premature aging. Due to inefficient decay, senescence-related transcripts like ORESARA1 (ORE1), SENESCENCE-ASSOCIATED GENE 21 (SAG21), NAC-like, activated by AP3/PI (NAP), and NONYELLOWING 1 (NYE1) over-accumulated in mta thereby causing accelerated senescence during DILS. Overall, our data propose that m6A modification is involved in regulating the biological response to senescence in plants, providing targets for engineering stress tolerance of crops.

The emerging role of epitranscriptome in shaping stress responses in plants.

RNA modifications and editing changes constitute 'epitranscriptome' and are crucial in regulating the development and stress response in plants. Exploration of the epitranscriptome and associated machinery would facilitate the engineering of stress tolerance in crops. RNA editing and modifications post-transcriptionally decorate almost all classes of cellular RNAs, including tRNAs, rRNAs, snRNAs, lncRNAs and mRNAs, with more than 170 known modifications, among which m6A, Ψ, m5C, 8-OHG and C-to-U editing are the most abundant. Together, these modifications constitute the "epitranscriptome", and contribute to changes in several RNA attributes, thus providing an additional structural and functional diversification to the "cellular messages" and adding another layer of gene regulation in organisms, including plants. Numerous evidences suggest that RNA modifications have a widespread impact on plant development as well as in regulating the response of plants to abiotic and biotic stresses. High-throughput sequencing studies demonstrate that the landscapes of m6A, m5C, Am, Cm, C-to-U, U-to-G, and A-to-I editing are remarkably dynamic during stress conditions in plants. GO analysis of transcripts enriched in Ψ, m6A and m5C modifications have identified bonafide components of stress regulatory pathways. Furthermore, significant alterations in the expression pattern of genes encoding writers, readers, and erasers of certain modifications have been documented when plants are grown in challenging environments. Notably, manipulating the expression levels of a few components of RNA editing machinery markedly influenced the stress tolerance in plants. We provide updated information on the current understanding on the contribution of RNA modifications in shaping the stress responses in plants. Unraveling of the epitranscriptome has opened new avenues for designing crops with enhanced productivity and stress resilience in view of global climate change.

"Millet Models" for harnessing nuclear factor-Y transcription factors to engineer stress tolerance in plants: current knowledge and emerging paradigms.

The main purpose of this review is to shed light on the role of millet models in imparting climate resilience and nutritional security and to give a concrete perspective on how NF-Y transcription factors can be harnessed for making cereals more stress tolerant. Agriculture faces significant challenges from climate change, bargaining, population, elevated food prices, and compromises with nutritional value. These factors have globally compelled scientists, breeders, and nutritionists to think of some options that can combat the food security crisis and malnutrition. To address these challenges, mainstreaming the climate-resilient and nutritionally unparalleled alternative crops like millet is a key strategy. The C4 photosynthetic pathway and adaptation to low-input marginal agricultural systems make millets a powerhouse of important gene and transcription factor families imparting tolerance to various kinds of biotic and abiotic stresses. Among these, the nuclear factor-Y (NF-Y) is one of the prominent transcription factor families that regulate diverse genes imparting stress tolerance. The primary purpose of this article is to shed light on the role of millet models in imparting climate resilience and nutritional security and to give a concrete perspective on how NF-Y transcription factors can be harnessed for making cereals more stress tolerant. Future cropping systems could be more resilient to climate change and nutritional quality if these practices were implemented.

Transcriptome meta-analysis of abiotic stresses-responsive genes and identification of candidate transcription factors for broad stress tolerance in wheat.

Under field conditions, wheat is subjected to single or multiple stress conditions. The elucidation of the molecular mechanism of stress response is a key step to identify candidate genes for stress resistance in plants. In this study, RNA-seq data analysis identified 17.324, 10.562, 5.510, and 8.653 differentially expressed genes (DEGs) under salt, drought, heat, and cold stress, respectively. Moreover, the comparison of DEGs from each stress revealed 2374 shared genes from which 40% showed highly conserved expression patterns. Moreover, co-expression network analysis and GO enrichment revealed co-expression modules enriched with genes involved in transcription regulation, protein kinase pathway, and genes responding to phytohormones or modulating hormone levels. The expression of 15 selected transcription factor encoding genes was analyzed under abiotic stresses and ABA treatment in durum wheat. The identified transcription factor genes are excellent candidates for genetic engineering of stress tolerance in wheat.

Integrated physiological and comparative proteomics analysis of contrasting genotypes of pearl millet reveals underlying salt-responsive mechanisms.

Salinity stress poses a significant risk to plant development and agricultural yield. Therefore, elucidation of stress-response mechanisms has become essential to identify salt-tolerance genes in plants. In the present study, two genotypes of pearl millet (Pennisetum glaucum L.) with contrasting tolerance for salinity exhibited differential morpho-physiological and proteomic responses under 150 mM NaCl. The genotype IC 325825 was shown to withstand the stress better than IP 17224. The salt-tolerance potential of IC 325825 was associated with its ability to maintain intracellular osmotic, ionic, and redox homeostasis and membrane integrity under stress. The IC 325825 genotype exhibited a higher abundance of C4 photosynthesis enzymes, efficient enzymatic and non-enzymatic antioxidant system, and lower Na+ /K+ ratio compared with IP 17224. Comparative proteomics analysis revealed greater metabolic perturbation in IP 17224 under salinity, in contrast to IC 325825 that harbored pro-active stress-responsive machinery, allowing its survival and better adaptability under salt stress. The differentially abundant proteins were in silico characterized for their functions, subcellular-localization, associated pathways, and protein-protein interaction. These proteins were mainly involved in photosynthesis/response to light stimulus, carbohydrate and energy metabolism, and stress responses. Proteomics data were validated through expression profiling of the selected genes, revealing a poor correlation between protein abundance and their relative transcript levels. This study has provided novel insights into salt adaptive mechanisms in P. glaucum, demonstrating the power of proteomics-based approaches. The critical proteins identified in the present study could be further explored as potential objects for engineering stress tolerance in salt-sensitive major crops.

Engineering cereal crops for enhanced abiotic stress tolerance

In the scenario of global climate change, abiotic stresses such as rising temperature, water deficit and stress combinations are posing serious threats to sustainable agricultural production, and these factors account for more yield losses than any other factor. Developing crop plants with enhanced abiotic stress tolerance has become a priority now-a-days. Agricultural biotechnology including genomics-assisted breeding and genetic engineering help in studying and understanding the complex nature of abiotic stress responses and provide measures for enhancing crop productivity under adverse environmental conditions. Plants respond and adapt to adverse environmental factors by activating molecular network cascades that are implicated in stress perception, signal transduction, genes expression and accumulation of certain metabolites. Various functional genomics approaches have helped to identify numerous genes involved in stress-associated molecular regulatory networks in both model plants and non-model crop species. Thus engineering genes that activate the transcription of other stress responsive genes or play important roles in protection and maintenance of cellular components and macromolecules might expedite crop improvement programs. Several of these candidate genes have been transformed in agriculturally important crops to improve their abiotic stress tolerance. Nonetheless, genetic engineering for stress tolerance not only encompasses attempts to engineer “single action genes” and “gene pyramiding” but also introgressions of regulatory machinery involving transcription factors. Further, development of transgenic crops not only depends upon the success rate of genetic transformation but also upon their proper integration and evaluation of stress tolerance. This review thus, summarizes the recent progress in the application of genetic engineering and/or transgenic technology for crop improvement in order to realize global food security by developing varieties superior in abiotic stress tolerance.

Ectopic expression of BrALDH7B2 gene encoding an antiquitin from Brassica rapa confers tolerance to abiotic stresses and improves photosynthetic performance under salt stress in tobacco

Improved plant performance under salt and drought stresses has been a challenging task to the plant due to the complexity and multitude of genes that govern them. We describe here, isolation and characterization of an aldehyde dehydrogenase gene (BrALDH7B2) that encodes an antiquitin isolated from Brassica rapa. Overexpression of the antiquitin gene in tobacco conferred tolerance to salt and drought stresses at the seedling stage as reflected by fresh weight, root length, and chlorophyll and carotenoid contents. The improved physiological characteristics observed in the transgenics may be ascribed to better PIabs, Pn, gs, WUE and E leading to better endurance under salt stress. The overexpression of BrALDH7B2 improved photosynthetic performance accompanied by reduced ROS formation followed by repressed cell-death in roots as determined by intracellular accumulation of H2DCF-DA and PI during salt stress in transgenic plants. Under salt stress, ALDH expressing tobacco transgenic plants showed activation of abiotic stress-responsive genes and vigorous ROS-scavenging system at the molecular level. Altogether, BrALDH7B2 represents a good candidate gene for orchestrating stress responses and with potential for engineering stress tolerance in different crops. To the best of our knowledge, this is the first report on functional study of BrALDH gene in the heterologous system tobacco with enhanced photosynthetic performance under salt stress.

Contrasting Responses to Stress Displayed by Tobacco Overexpressing an Algal Plastid Terminal Oxidase in the Chloroplast.

The plastid terminal oxidase (PTOX) – an interfacial diiron carboxylate protein found in the thylakoid membranes of chloroplasts – oxidizes plastoquinol and reduces molecular oxygen to water. It is believed to play a physiologically important role in the response of some plant species to light and salt (NaCl) stress by diverting excess electrons to oxygen thereby protecting photosystem II (PSII) from photodamage. PTOX is therefore a candidate for engineering stress tolerance in crop plants. Previously, we used chloroplast transformation technology to over express PTOX1 from the green alga Chlamydomonas reinhardtii in tobacco (generating line Nt-PTOX-OE). Contrary to expectation, growth of Nt-PTOX-OE plants was more sensitive to light stress. Here we have examined in detail the effects of PTOX1 on photosynthesis in Nt-PTOX-OE tobacco plants grown at two different light intensities. Under ‘low light’ (50 μmol photons m–2 s–1) conditions, Nt-PTOX-OE and WT plants showed similar photosynthetic activities. In contrast, under ‘high light’ (125 μmol photons m–2 s–1) conditions, Nt-PTOX-OE showed less PSII activity than WT while photosystem I (PSI) activity was unaffected. Nt-PTOX-OE grown under high light also failed to increase the chlorophyll a/b ratio and the maximum rate of CO2 assimilation compared to low-light grown plants, suggesting a defect in acclimation. In contrast, Nt-PTOX-OE plants showed much better germination, root length, and shoot biomass accumulation than WT when exposed to high levels of NaCl and showed better recovery and less chlorophyll bleaching after NaCl stress when grown hydroponically. Overall, our results strengthen the link between PTOX and the resistance of plants to salt stress.

The Q-System as a Synthetic Transcriptional Regulator in Plants.

A primary focus of the rapidly growing field of plant synthetic biology is to develop technologies to precisely regulate gene expression and engineer complex genetic circuits into plant chassis. At present, there are few orthogonal tools available for effectively controlling gene expression in plants, with most researchers instead using a limited set of viral elements or truncated native promoters. A powerful repressible-and engineerable-binary system that has been repurposed in a variety of eukaryotic systems is the Q-system from Neurospora crassa. Here, we demonstrate the functionality of the Q-system in plants through transient expression in soybean (Glycine max) protoplasts and agroinfiltration in Nicotiana benthamiana leaves. Further, using functional variants of the QF transcriptional activator, it was possible to modulate the expression of reporter genes and to fully suppress the system through expression of the QS repressor. As a potential application for plant-based biosensors (phytosensors), we demonstrated the ability of the Q-system to amplify the signal from a weak promoter, enabling remote detection of a fluorescent reporter that was previously undetectable. In addition, we demonstrated that it was possible to coordinate the expression of multiple genes through the expression of a single QF activator. Based on the results from this study, the Q-system represents a powerful orthogonal tool for precise control of gene expression in plants, with envisioned applications in metabolic engineering, phytosensors, and biotic and abiotic stress tolerance.

Heterologous Expression of Three Ammopiptanthus mongolicus Dehydrin Genes Confers Abiotic Stress Tolerance in Arabidopsis thaliana

Ammopiptanthus mongolicus, a xerophyte plant that belongs to the family Leguminosae, adapts to extremely arid, hot, and cold environments, making it an excellent woody plant to study the molecular mechanisms underlying abiotic stress tolerance. Three dehydrin genes, AmDHN132, AmDHN154, and AmDHN200 were cloned from abiotic stress treated A. mongolicus seedlings. Cytomembrane-located AmDHN200, nucleus-located AmDHN154, and cytoplasm and nucleus-located AmDHN132 were characterized by constitutive overexpression of their genes in Arabidopsis thaliana. Overexpression of AmDHN132, AmDHN154, and AmDHN200 in transgenic Arabidopsis improved salt, osmotic, and cold tolerances, with AmDHN132 having the largest effect, whereas the growth of transformed plants is not negatively affected. These results indicate that AmDHNs contribute to the abiotic stress tolerance of A. mongolicus and that AmDHN genes function differently in response to abiotic stresses. Furthermore, they have the potential to be used in the genetic engineering of stress tolerance in higher plants.

From laboratory to field: yield stability and shade avoidance genes are massively differentially expressed in the field.

To unravel molecular mechanisms with the ultimate goal to achieve improved stress resilience or increased yield, plants are often studied under highly controlled conditions in which stresses are applied and in which growth‐ or architecture‐related traits are meticulously recorded. Over the past decades, this has led to a boost in our understanding of key molecular players and in strategies to improve yield stability. However, many single‐gene traits fail to translate into applications (Nuccio et al., 2018).

Exposure to High-Intensity Light Systemically Induces Micro-Transcriptomic Changes in Arabidopsis thaliana Roots.

In full sunlight, plants often experience a light intensity exceeding their photosynthetic capacity and causing the activation of a set of photoprotective mechanisms. Numerous reports have explained, on the molecular level, how plants cope with light stress locally in photosynthesizing leaves; however, the response of below-ground organs to above-ground perceived light stress is still largely unknown. Since small RNAs are potent integrators of multiple processes including stress responses, here, we focus on changes in the expression of root miRNAs upon high-intensity-light (HL) stress. To achieve this, we used Arabidopsis thaliana plants growing in hydroponic conditions. The expression of several genes that are known as markers of redox changes was examined over time, with the results showing that typical HL stress signals spread to the below-ground organs. Additionally, micro-transcriptomic analysis of systemically stressed roots revealed a relatively limited reaction, with only 17 up-regulated and five down-regulated miRNAs. The differential expression of candidates was confirmed by RT-qPCR. Interestingly, the detected differences in miRNA abundance disappeared when the roots were separated from the shoots before HL treatment. Thus, our results show that the light stress signal is induced in rosettes and travels through the plant to affect root miRNA levels. Although the mechanism of this regulation is unknown, the engagement of miRNA may create a regulatory platform orchestrating adaptive responses to various simultaneous stresses. Consequently, further research on systemically HL-regulated miRNAs and their respective targets has the potential to identify attractive sequences for engineering stress tolerance in plants.

Transcriptome analysis reveals regulatory framework for salt and osmotic tolerance in a succulent xerophyte

BackgroundZygophyllum xanthoxylum is a succulent xerophyte with remarkable tolerance to diverse abiotic stresses. Previous studies have revealed important physiological mechanisms and identified functional genes associated with stress tolerance. However, knowledge of the regulatory genes conferring stress tolerance in this species is poorly understood.ResultsHere, we present a comprehensive analysis of regulatory genes based on the transcriptome of Z. xanthoxylum roots exposed to osmotic stress and salt treatments. Significant changes were observed in transcripts related to known and obscure stress-related hormone signaling pathways, in particular abscisic acid and auxin. Significant changes were also found among key classes of early response regulatory genes encoding protein kinases, transcription factors, and ubiquitin-mediated proteolysis machinery. Network analysis shows a highly integrated matrix formed by these conserved and novel gene products associated with osmotic stress and salt in Z. xanthoxylum. Among them, two previously uncharacterized NAC (NAM/ATAF/CUC) transcription factor genes, ZxNAC083 (Unigene16368_All) and ZxNAC035 (CL6534.Contig1_All), conferred tolerance to salt and drought stress when constitutively overexpressed in Arabidopsis plants.ConclusionsThis study provides a unique framework for understanding osmotic stress and salt adaptation in Z. xanthoxylum including novel gene targets for engineering stress tolerance in susceptible crop species.

Концепция динамического психобиологического гомеостаза и полиморфизм стрессоустойчивости как новый взгляд в психологии стресса

The article presents the results of a theoretical study of stress resistance, based on the analysis of modern ideas of foreign psychology. The aim of the study was to identify modern perspective approaches to the study of stress resistance for further interdisciplinary impacts. The characteristics of two types of stress tolerance ˗ “engineering” and “environmental” are presented. “Engineering stress tolerance” is associated with the restoration of the initial state of the body after the experienced stress. “Environmental stress tolerance” is characterized by the transition of the body to an alternative state. Explaining the existence of two forms of stress resistance opens up new perspectives in studies of stress psychology.

Application of proteomics and metabolomics in microbial metabolic engineering

Construction of microbial cell factories is a feasible strategy for the sustainable production of chemicals, biofuels, and pharmaceutical molecules. However, the complex metabolism and rigid regulation of microbes hinders the efficient synthesis of the products of interest. Proteomics and metabolomics analyze enzymes and metabolites in terms of systems biology, thus unraveling complex biological systems and providing significant clues for microbial metabolic engineering. Here, the applications of proteomics and metabolomics in microbial metabolic engineering were reviewed, including the construction of genome-scale metabolic models, optimization of microbial product synthesis, guidance of microbial stress tolerance engineering, and prediction of rate-limiting steps. In addition, proteomics and metabolomics could be employed to explore secondary metabolic pathways in plants, to reveal novel genes or pathways for microbial synthesis of natural products. Finally, the development of bio-big data was discussed.

Plant small RNAs: the essential epigenetic regulators of gene expression for salt-stress responses and tolerance.

Saline environment cues distort the plant growth, development and crop yield. Epigenetics has emerged as one of the prime themes in plant functional genomics for molecular-stress-physiology research, as copious studies have provided new visions into the epigenetic control of stress adaptations. The epigenetic control is associated with the regulation of the expression of stress-related genes which also comprises many steady alterations inherited in next cellular generation as stress memory. These epigenetic amendments also implicate induction of small RNA (sRNA)-mediated fine-tuning of transcriptional and post-transcriptional regulations of gene expression. These tiny (19-24nt) RNA species, particularly microRNAs (miRNAs) besides endogenous small interfering RNA (siRNA) have emerged as important responsive entities for epigenetic modulation of salt-stress effects on plants. There is a recent upsurge in development of tools and databases useful for prediction, identification and validation of small RNAs (sRNAs) and their target messenger RNAs (mRNAs). Therefore, these small but key regulatory molecules have received a wide attention in post-genomic era as potential targets for engineering stress tolerance in major glycophytic crops, though it is yet to be explored optimally. This review aims to provide critical updates on plant sRNAs as key epigenetic regulators of plant salt-stress responses, their target prediction and validation, computational tools and databases available for plant small RNAs, besides discussing their roles in salt-stress regulatory networks and adaptive mechanisms in plants, with special emphasis on their exploration for engineering salinity tolerance in plants.

RECoN: Rice Environment Coexpression Network for Systems Level Analysis of Abiotic-Stress Response.

Transcriptional profiling is a prevalent and powerful approach for capturing the response of crop plants to environmental stresses, e.g., response of rice to drought. However, functionally interpreting the resulting genome-wide gene expression changes is severely hampered by the large gaps in our genomic knowledge about which genes work together in cellular pathways/processes in rice. Here, we present a new web resource – RECoN – that relies on a network-based approach to go beyond currently limited annotations in delineating functional and regulatory perturbations in new rice transcriptome datasets generated by a researcher. To build RECoN, we first enumerated 1,744 abiotic stress-specific gene modules covering 28,421 rice genes (>72% of the genes in the genome). Each module contains a group of genes tightly coexpressed across a large number of environmental conditions and, thus, is likely to be functionally coherent. When a user provides a new differential expression profile, RECoN identifies modules substantially perturbed in their experiment and further suggests deregulated functional and regulatory mechanisms based on the enrichment of current annotations within the predefined modules. We demonstrate the utility of this resource by analyzing new drought transcriptomes of rice in three developmental stages, which revealed large-scale insights into the cellular processes and regulatory mechanisms involved in common and stage-specific drought responses. RECoN enables biologists to functionally explore new data from all abiotic stresses on a genome-scale and to uncover gene candidates, including those that are currently functionally uncharacterized, for engineering stress tolerance.

Transgenic approaches to enhance salt and drought tolerance in plants

Abiotic stresses including drought, salinity, extreme temperatures, and heavy metal stress, are responsible for reduction in crop yields around the globe. The situation may further worsen with the reduction in arable land in countries of Asia due to population explosion. Though the conventional breeding programs developed stress tolerant varieties, the pace was very slow due to complication in abiotic stress by complex multigene traits and cumbersome phenotyping procedures. With the dawn of plant biotechnology, enormous efforts have been made to engineer stress tolerance in major-crops and model-plants. A substantial number of stress-responsive and/or stress-regulated genes have been identified. Addressing these constraints would help us in better understanding how transgenic plants are tolerating drought and salinity. Overview of the present and future methodologies would enable us to gain more insights in understanding transgenic approaches for tolerance to abiotic stress.

Dehydration responsive element binding transcription factors and their applications for the engineering of stress tolerance.

Dehydration responsive element binding (DREB) factors or CRT element binding factors (CBFs) are members of the AP2/ERF family, which comprises a large number of stress-responsive regulatory genes. This review traverses almost two decades of research, from the discovery of DREB/CBF factors to their optimization for application in plant biotechnology. In this review, we describe (i) the discovery, classification, structure, and evolution of DREB genes and proteins; (ii) induction of DREB genes by abiotic stresses and involvement of their products in stress responses; (iii) protein structure and DNA binding selectivity of different groups of DREB proteins; (iv) post-transcriptional and post-translational mechanisms of DREB transcription factor (TF) regulation; and (v) physical and/or functional interaction of DREB TFs with other proteins during plant stress responses. We also discuss existing issues in applications of DREB TFs for engineering of enhanced stress tolerance and improved performance under stress of transgenic crop plants.