CRISPR/Cas9-Mediated Gene Editing in Grain Crops

The development of reliable and efficient techniques for making precise targeted changes in the genome of living organisms has been a long-standing objective of researchers throughout the world. In plants, different methods, each with several different variations, have been developed for this purpose, though many of them are hampered either by providing only temporary modification of gene function or unpredictable off-target results. The recent discovery of clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated 9 (Cas9) nucleases started a new era in genome editing. Basically, the CRISPR/Cas system is a natural immune response of prokaryotes to resist foreign genetic elements entering via plasmids and phages. Through this naturally occurring gene editing system, bacteria create DNA segments known as CRISPR arrays that allow them to “remember” foreign genetic material for protection against it and other similar sequences in the future. This system has now been adopted by researchers in laboratory to create a short guide RNA that binds to specific target sequences of DNA in eukaryotic genome, and the Cas9 enzyme cuts the DNA at the targeted location. Once cut, the cell’s endogenous DNA repair machinery is used to add, delete, or replace pieces of genetic material. Though CRISPR/Cas9 technology has been recently developed, it has started to be regularly used for gene editing in plants as well as animals to good success. It has been proved as an efficient transgene-free technique. A simple search on PubMed (NCBI) shows that among all plants, 80 different studies published since 2013 involved CRISPR/Cas9-mediated genome editing in rice. Of these, 20, 13, and 24 papers have been published in 2019, 2018, and 2017, respectively. Furthermore, 20 different studies published since 2014 utilized CRISPR/Cas9 system for gene editing in wheat, where five of these studies were published in 2019 and seven were published in 2018. Genomes of other grain crops edited through this technique include maize, sorghum, barley, etc. This indicates the high utility of this technique for gene editing in grain crops. Here we emphasize on CRISPR/Cas9-mediated gene editing in rice, wheat, and maize

Seed priming with different agents mitigate alkalinity induced oxidative damage and improves maize growth

Soil alkalinity is a severe threat to crop production globally as it markedly retards plant growth. Different techniques are used to mitigate alkaline stress, but priming techniques are considered the most appropriate. The current study was carried out in complete randomized design (CRD) to evaluate the effect of different priming techniques on maize crop grown under different levels of alkalinity stress. The experiment was comprised of different treatments of alkalinity stress (AS) including, control, 6 dS m-1 and 12 dS m-1 and different priming techniques including control, hydro-priming (HP), osmo-priming (OP) with potassium nitrate: KNO3) and redox-priming (RP) with hydrogen peroxide (H2O2). Results indicated that alkalinity stress significantly reduced plant growth and biomass production and induced severe alterations in physiological attributes and antioxidant activities. Soil alkalinity significantly reduced the root and shoot growth and subsequent biomass production by increasing electrolyte leakage (70.60%), hydrogen peroxide (H2O2: 31.65%), malondialdehyde (MDA: 46.23%) and sodium (Na+) accumulation (22.76%) and reduction in photosynthetic pigments, relative water contents (RWC), total soluble proteins (TSP) and free amino acids, potassium (K+) accumulation. However, priming treatments significantly alleviated the alkalinity-induced toxic effects and improved plant growth. OP (KNO3) remained the top performing. It appreciably improved plant growth owing to the improved synthesis of photosynthetic pigments, better RWC (16.42%), TSP (138.28%), FAA (178.37%), and K+ accumulation (31.385) and improved antioxidant activities (APX and CAT) by favoring the Na+ exclusion and maintenance of optimum Na+/K+. In conclusion, KNO3 priming is an imperative seed priming practice to improve maize growth and biomass production under alkalinity stress

Transcription Factors as the "Blitzkrieg" of Plant Defense : A Pragmatic View of Nitric Oxide's Role in Gene Regulation

Plants are in continuous conflict with the environmental constraints and their sessile nature demands a fine-tuned, well-designed defense mechanism that can cope with a multitude of biotic and abiotic assaults. Therefore, plants have developed innate immunity, R-gene-mediated resistance, and systemic acquired resistance to ensure their survival. Transcription factors (TFs) are among the most important genetic components for the regulation of gene expression and several other biological processes. They bind to specific sequences in the DNA called transcription factor binding sites (TFBSs) that are present in the regulatory regions of genes. Depending on the environmental conditions, TFs can either enhance or suppress transcriptional processes. In the last couple of decades, nitric oxide (NO) emerged as a crucial molecule for signaling and regulating biological processes. Here, we have overviewed the plant defense system, the role of TFs in mediating the defense response, and that how NO can manipulate transcriptional changes including direct post-translational modifications of TFs. We also propose that NO might regulate gene expression by regulating the recruitment of RNA polymerase during transcription

Endophyte-mediated stress tolerance in plants : a sustainable strategy to enhance resilience and assist crop improvement

Biotic and abiotic stresses severely affect agriculture by affecting crop productivity, soil fertility, and health. These stresses may have significant financial repercussions, necessitating a practical, cost-effective, and ecologically friendly approach to lessen their negative impacts on plants. Several agrochemicals, such as fertilizers, pesticides, and insecticides, are used to improve plant health and protection; however, these chemical supplements have serious implications for human health. Plants being sessile cannot move or escape to avoid stress. Therefore, they have evolved to develop highly beneficial interactions with endophytes. The targeted use of beneficial plant endophytes and their role in combating biotic and abiotic stresses are gaining attention. Therefore, it is important to experimentally validate these interactions and determine how they affect plant fitness. This review highlights research that sheds light on how endophytes help plants tolerate biotic and abiotic stresses through plant–symbiont and plant–microbiota interactions. There is a great need to focus research efforts on this vital area to achieve a system-level understanding of plant–microbe interactions that occur naturally

Salinity Stress-Mediated Suppression of Expression of Salt Overly Sensitive Signaling Pathway Genes Suggests Negative Regulation by AtbZIP62 Transcription Factor in Arabidopsis thaliana

Salt stress is one of the most serious threats in plants, reducing crop yield and production. The salt overly sensitive (SOS) pathway in plants is a salt-responsive pathway that acts as a janitor of the cell to sweep out Na+ ions. Transcription factors (TFs) are key regulators of expression and/or repression of genes. The basic leucine zipper (bZIP) TF is a large family of TFs regulating various cellular processes in plants. In the current study, we investigated the role of the Arabidopsis thaliana bZIP62 TF in the regulation of SOS signaling pathway by measuring the transcript accumulation of its key genes such as SOS1, 2, and 3, in both wild-type (WT) and atbzip62 knock-out (KO) mutants under salinity stress. We further observed the activation of enzymatic and non-enzymatic antioxidant systems in the wild-type, atbzip62, atcat2 (lacking catalase activity), and atnced3 (lacking 9-cis-epoxycarotenoid dioxygenase involved in the ABA pathway) KO mutants. Our findings revealed that atbzip62 plants exhibited an enhanced salt-sensitive phenotypic response similar to atnced3 and atcat2 compared to WT, 10 days after 150 mM NaCl treatment. Interestingly, the transcriptional levels of SOS1, SOS2, and SOS3 increased significantly over time in the atbzip62 upon NaCl application, while they were downregulated in the wild type. We also measured chlorophyll a and b, pheophytin a and b, total pheophytin, and total carotenoids. We observed that the atbzip62 exhibited an increase in chlorophyll and total carotenoid contents, as well as proline contents, while it exhibited a non-significant increase in catalase activity. Our results suggest that AtbZIP62 negatively regulates the transcriptional events of SOS pathway genes, AtbZIP18 and AtbZIP69 while modulating the antioxidant response to salt tolerance in Arabidopsis

Abiotic stress in plants, stress perception to molecular response and role of biotechnological tools in stress resistance

Plants, due to their sessile nature, face several environmental adversities. Abiotic stresses such as heat, cold, drought, heavy metals, and salinity are serious threats to plant production and yield. To cope with these stresses, plants have developed sophisticated mechanisms to avoid or resist stress conditions. A proper response to abiotic stress depends primarily on how plants perceive the stress signal, which in turn leads to initiation of signaling cascades and induction of resistance genes. New biotechnological tools such as RNA-seq and CRISPR-cas9 are quite useful in identifying target genes on a global scale, manipulating these genes to achieve tolerance, and helping breeders to develop stress-tolerant cultivars. In this review, we will briefly discuss the adverse effects of key abiotic stresses such as cold, heat, drought, and salinity. We will also discuss how plants sense various stresses and the importance of biotechnological tools in the development of stress-tolerant cultivars

Comprehensive Analyses of Nitric Oxide-Induced Plant Stem Cell-Related Genes in Arabidopsis thaliana

Plant stem cells are pluripotent cells that have diverse applications in regenerative biology and medicine. However, their roles in plant growth and disease resistance are often overlooked. Using high-throughput RNA-seq data, we identified approximately 20 stem cell-related differentially expressed genes (DEGs) that were responsive to the nitric oxide (NO) donor S-nitrosocysteine (CySNO) after six hours of infiltration. Among these DEGs, the highest number of positive correlations (R ≥ 0.8) was observed for CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) 12. Gene ontology (GO) terms for molecular function showed DEGs associated with signal transduction and receptor activity. A promoter study of these DEGs showed the presence of cis-acting elements that are involved in growth as well as the regulation of abiotic and biotic stress. Phylogenetic analysis of the Arabidopsis stem cell-related genes and their common orthologs in rice, soybean, poplar, and tomato suggested that most soybean stem cell-related genes were grouped with the Arabidopsis CLE type of stem cell genes, while the rice stem cell-related genes were grouped with the Arabidopsis receptor-like proteins. The functional genomic-based characterization of the role of stem cell DEGs showed that under control conditions, the clv1 mutant showed a similar phenotype to that of the wild-type (WT) plants; however, under CySNO-mediated nitrosative stress, clv1 showed increased shoot and root length compared to WT. Furthermore, the inoculation of clv1 with virulent Pst DC3000 showed a resistant phenotype with fewer pathogens growing at early time points. The qRT-PCR validation and correlation with the RNA-seq data showed a Pearson correlation coefficient of >0.8, indicating the significantly high reliability of the RNA-seq analysis

Pseudomonas syringae infectivity correlates to altered transcript and metabolite levels of Arabidopsis mediator mutants

Rapid metabolic responses to pathogens are essential for plant survival and depend on numerous transcription factors. Mediator is the major transcriptional co-regulator for integration and transmission of signals from transcriptional regulators to RNA polymerase II. Using four Arabidopsis Mediator mutants, med16, med18, med25 and cdk8, we studied how differences in regulation of their transcript and metabolite levels correlate to their responses to Pseudomonas syringae infection. We found that med16 and cdk8 were susceptible, while med25 showed increased resistance. Glucosinolate, phytoalexin and carbohydrate levels were reduced already before infection in med16 and cdk8, but increased in med25, which also displayed increased benzenoids levels. Early after infection, wild type plants showed reduced glucosinolate and nucleoside levels, but increases in amino acids, benzenoids, oxylipins and the phytoalexin camalexin. The Mediator mutants showed altered levels of these metabolites and in regulation of genes encoding key enzymes for their metabolism. At later stage, mutants displayed defective levels of specific amino acids, carbohydrates, lipids and jasmonates which correlated to their infection response phenotypes. Our results reveal that MED16, MED25 and CDK8 are required for a proper, coordinated transcriptional response of genes which encode enzymes involved in important metabolic pathways for Arabidopsis responses to Pseudomonas syringae infections

Nitric oxide mediated transcriptome profiling reveals activation of multiple regulatory pathways in Arabidopsis thaliana

Imbalance between the accumulation and removal of nitric oxide and its derivatives is a challenge faced by all plants at the cellular level, and is especially important under stress conditions. Exposure of plants to various biotic and abiotic stresses causes rapid changes in cellular redox tone potentiated by the rise in reactive nitrogen species that serve as signaling molecules in mediating defensive responses. To understand mechanisms mediated by these signaling molecules, we performed a large-scale analysis of the Arabidopsis transcriptome induced by nitrosative stress. We generated an average of 84 and 91 million reads from three replicates each of control and 1 mM S-nitrosocysteine (CysNO)-infiltrated Arabidopsis leaf samples, respectively. After alignment, more than 95% of all reads successfully mapped to the reference and 32,535 genes and 55,682 transcripts were obtained. CysNO infiltration caused differential expression of 6436 genes (3448 up-regulated and 2988 down-regulated) and 6214 transcripts (3335 up-regulated and 2879 down-regulated) 6 h post-infiltration. These differentially expressed genes were found to be involved in key physiological processes, including plant defense against various biotic and abiotic stresses, hormone signaling, and other developmental processes. After quantile normalization of the FPKM values followed by student's T-test (P < 0.05) we identified 1165 DEGs (463 up-regulated and 702 down-regulated) with at least 2-folds change in expression after CysNO treatment. Expression patterns of selected genes involved in various biological pathways were verified using quantitative real-time PCR. This study provides comprehensive information about plant responses to nitrosative stress at transcript level and would prove helpful in understanding and incorporating mechanisms associated with nitrosative stress responses in plants