C-Type LECTIN receptor-like kinase 1 and ACTIN DEPOLYMERIZING FACTOR 3 are key components of plasmodesmata callose modulation.

Plasmodesmata (PDs) are intercellular organelles carrying multiple membranous nanochannels that allow the trafficking of cellular signalling molecules. The channel regulation of PDs occurs dynamically and is required in various developmental and physiological processes. It is well known that callose is a critical component in regulating PD permeability or symplasmic connectivity, but the understanding of the signalling pathways and mechanisms of its regulation is limited. Here, we used the reverse genetic approach to investigate the role of C-type lectin receptor-like kinase 1 (CLRLK1) in the aspect of PD callose-modulated symplasmic continuity. Here, we found that loss-of-function mutations in CLRLK1 resulted in excessive PD callose deposits and reduced symplasmic continuity, resulting in an accelerated gravitropic response. The protein interactome study also found that CLRLK1 interacted with actin depolymerizing factor 3 (ADF3) in vitro and in plants. Moreover, mutations in ADF3 result in elevated PD callose deposits and faster gravitropic response. Our results indicate that CLRLK1 and ADF3 negatively regulate PD callose accumulation, contributing to fine-tuning symplasmic opening apertures. Overall, our studies identified two key components involved in the deposits of PD callose and provided new insights into how symplasmic connectivity is maintained by the control of PD callose homoeostasis.

Enhancement of specialized metabolites using CRISPR/Cas gene editing technology in medicinal plants.

Plants are the richest source of specialized metabolites. The specialized metabolites offer a variety of physiological benefits and many adaptive evolutionary advantages and frequently linked to plant defense mechanisms. Medicinal plants are a vital source of nutrition and active pharmaceutical agents. The production of valuable specialized metabolites and bioactive compounds has increased with the improvement of transgenic techniques like gene silencing and gene overexpression. These techniques are beneficial for decreasing production costs and increasing nutritional value. Utilizing biotechnological applications to enhance specialized metabolites in medicinal plants needs characterization and identification of genes within an elucidated pathway. The breakthrough and advancement of CRISPR/Cas-based gene editing in improving the production of specific metabolites in medicinal plants have gained significant importance in contemporary times. This article imparts a comprehensive recapitulation of the latest advancements made in the implementation of CRISPR-gene editing techniques for the purpose of augmenting specific metabolites in medicinal plants. We also provide further insights and perspectives for improving metabolic engineering scenarios in medicinal plants.

Generation of parthenocarpic tomato plants in multiple elite cultivars using the CRISPR/Cas9 system.

Tomato (Solanum lycopersicum L.) is one of the most important crops in the world for its fruit production. Advances in cutting-edge techniques have enabled the development of numerous critical traits related to the quality and quantity of tomatoes. Genetic engineering techniques, such as gene transformation and gene editing, have emerged as powerful tools for generating new plant varieties with superior traits. In this study, we induced parthenocarpic traits in a population of elite tomato (ET) lines. At first, the adaptability of ET lines to genetic transformation was evaluated to identify the best-performing lines by transforming the SlANT1 gene overexpression cassette and then later used to produce the SlIAA9 knockout lines using the CRISPR/Cas9 system. ET5 and ET8 emerged as excellent materials for these techniques and showed higher efficiency. Typical phenotypes of knockout sliaa9 were clearly visible in G0 and G1 plants, in which simple leaves and parthenocarpic fruits were observed. The high efficiency of the CRISPR/Cas9 system in developing new tomato varieties with desired traits in a short period was demonstrated by generating T-DNA-free homozygous sliaa9 knockout plants in the G1 generation. Additionally, a simple artificial fertilization method was successfully applied to recover seed production from parthenocarpic plants, securing the use of these varieties as breeding materials. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01452-1.

Prime editing: Mechanism insight and recent applications in plants.

Prime editing (PE) technology utilizes an extended prime editing guide RNA (pegRNA) to direct a fusion peptide consisting of nCas9 (H840) and reverse transcriptase (RT) to a specific location in the genome. This enables the installation of base changes at the targeted site using the extended portion of the pegRNA through RT activity. The resulting product of the RT reaction forms a 3' flap, which can be incorporated into the genomic site through a series of biochemical steps involving DNA repair and synthesis pathways. PE has demonstrated its effectiveness in achieving almost all forms of precise gene editing, such as base conversions (all types), DNA sequence insertions and deletions, chromosomal translocation and inversion and long DNA sequence insertion at safe harbour sites within the genome. In plant science, PE could serve as a groundbreaking tool for precise gene editing, allowing the creation of desired alleles to improve crop varieties. Nevertheless, its application has encountered limitations due to efficiency constraints, particularly in dicotyledonous plants. In this review, we discuss the step-by-step mechanism of PE, shedding light on the critical aspects of each step while suggesting possible solutions to enhance its efficiency. Additionally, we present an overview of recent advancements and future perspectives in PE research specifically focused on plants, examining the key technical considerations of its applications.

CRISPR base editor-based targeted random mutagenesis (BE-TRM) toolbox for directed evolution.

Directed evolution (DE) of desired locus by targeted random mutagenesis (TRM) tools is a powerful approach for generating genetic variations with novel or improved functions, particularly in complex genomes. TRM-based DE involves developing a mutant library of targeted DNA sequences and screening the variants for the desired properties. However, DE methods have for a long time been confined to bacteria and yeasts. Lately, CRISPR/Cas and DNA deaminase-based tools that circumvent enduring barriers such as longer life cycle, small library sizes, and low mutation rates have been developed to facilitate DE in native genetic environments of multicellular organisms. Notably, deaminase-based base editing-TRM (BE-TRM) tools have greatly expanded the scope and efficiency of DE schemes by enabling base substitutions and randomization of targeted DNA sequences. BE-TRM tools provide a robust platform for the continuous molecular evolution of desired proteins, metabolic pathway engineering, creation of a mutant library of desired locus to evolve novel functions, and other applications, such as predicting mutants conferring antibiotic resistance. This review provides timely updates on the recent advances in BE-TRM tools for DE, their applications in biology, and future directions for further improvements. [BMB Reports 2024; 57(1): 30-39].

CRISPR-Cas-mediated unfolded protein response control for enhancing plant stress resistance.

Plants consistently encounter environmental stresses that negatively affect their growth and development. To mitigate these challenges, plants have developed a range of adaptive strategies, including the unfolded protein response (UPR), which enables them to manage endoplasmic reticulum (ER) stress resulting from various adverse conditions. The CRISPR-Cas system has emerged as a powerful tool for plant biotechnology, with the potential to improve plant tolerance and resistance to biotic and abiotic stresses, as well as enhance crop productivity and quality by targeting specific genes, including those related to the UPR. This review highlights recent advancements in UPR signaling pathways and CRISPR-Cas technology, with a particular focus on the use of CRISPR-Cas in studying plant UPR. We also explore prospective applications of CRISPR-Cas in engineering UPR-related genes for crop improvement. The integration of CRISPR-Cas technology into plant biotechnology holds the promise to revolutionize agriculture by producing crops with enhanced resistance to environmental stresses, increased productivity, and improved quality traits.

Unlocking secrets of nature's chemists: Potential of CRISPR/Cas-based tools in plant metabolic engineering for customized nutraceutical and medicinal profiles.

Plant species have evolved diverse metabolic pathways to effectively respond to internal and external signals throughout their life cycle, allowing adaptation to their sessile and phototropic nature. These pathways selectively activate specific metabolic processes, producing plant secondary metabolites (PSMs) governed by genetic and environmental factors. Humans have utilized PSM-enriched plant sources for millennia in medicine and nutraceuticals. Recent technological advances have significantly contributed to discovering metabolic pathways and related genes involved in the biosynthesis of specific PSM in different food crops and medicinal plants. Consequently, there is a growing demand for plant materials rich in nutrients and bioactive compounds, marketed as "superfoods". To meet the industrial demand for superfoods and therapeutic PSMs, modern methods such as system biology, omics, synthetic biology, and genome editing (GE) play a crucial role in identifying the molecular players, limiting steps, and regulatory circuitry involved in PSM production. Among these methods, clustered regularly interspaced short palindromic repeats-CRISPR associated protein (CRISPR/Cas) is the most widely used system for plant GE due to its simple design, flexibility, precision, and multiplexing capabilities. Utilizing the CRISPR-based toolbox for metabolic engineering (ME) offers an ideal solution for developing plants with tailored preventive (nutraceuticals) and curative (therapeutic) metabolic profiles in an ecofriendly way. This review discusses recent advances in understanding the multifactorial regulation of metabolic pathways, the application of CRISPR-based tools for plant ME, and the potential research areas for enhancing plant metabolic profiles.

Profiling of conserved orthologs and miRNAs for understanding their role in salt tolerance mechanism of Sesuvium portulacastrum L.

BACKGROUND: Sesuvium portulacastrum is a facultative halophyte capable of thriving in a saline environment. Despite molecular studies conducted to unravel its salt adaptation mechanism, there is a paucity of information on the role of salt-responsive orthologs and microRNAs (miRNAs) in this halophyte. Here, we searched the orthology to identify salt-responsive orthologs and miRNA targets of Sesuvium using the Arabidopsis genome. METHODS: The relative fold change of orthologs, conserved miRNAs, and miRNA targets of Sesuvium was analyzed under 100 mM (LS) and 250 mM NaCl (HS) treatment at 24 h using qRT-PCR. The comparison between the expression of Sesuvium orthologs and Arabidopsis orthologs (Arabidopsis eFP browser database) was used to identify differentially expressed genes. RESULTS: Upon salt treatment, we found that SpCIPK3 (1.95-fold in LS and 2.90-fold in HS) in Sesuvium roots, and SpNHX7 (1.61-fold in LS and 6.39-fold in HS) and, SpSTPK2 (2.54-fold in LS and 7.65-fold in HS) in Sesuvium leaves were upregulated in a salt concentration-specific manner. In Arabidopsis, these genes were either downregulated or did not show significant variation, implicating its significance in the halophytic nature of Sesuvium. Furthermore, miRNAs like miR394a, miR396a, and miR397a exhibited a negative correlation with their targets-Frigida interacting protein 1, Cysteine proteinases superfamily protein, and Putative laccase, respectively under different salt treatments. CONCLUSION: The study revealed that the high salt tolerance in Sesuvium is associated with distinct transcriptional reprogramming, hence, to gain holistic mechanistic insights, global-scale profiling is required.

CRISPR-Cas9-based precise engineering of SlHyPRP1 protein towards multi-stress tolerance in tomato.

Recently, CRISPR-Cas9-based genome editing has been widely used for plant breeding. In our previous report, a tomato gene encoding hybrid proline-rich protein 1 (HyPRP1), a negative regulator of salt stress responses, has been edited using a CRISPR-Cas9 multiplexing approach that resulted in precise eliminations of its functional domains, proline-rich domain (PRD) and eight cysteine-motif (8CM). We subsequently demonstrated that eliminating the PRD domain of HyPRP1 in tomatoes conferred the highest level of salinity tolerance. In this study, we characterized the edited lines under several abiotic and biotic stresses to examine the possibility of multiple stress tolerance. Our data reveal that the 8CM removal variants of HK and the KO alleles of both HK and 15T01 cultivars exhibited moderate heat stress tolerance. Similarly, plants carrying either the domains of the PRD removal variant (PR1v1) or 8CM removal variants (PR2v2 and PR2v3) showed better germination under osmosis stress (up to 200 mM mannitol) compared to the WT control. Moreover, the PR1v1 line continuously grew after 5 days of water cutoff. When the edited lines were challenged with pathogenic bacteria of Pseudomonas syringae pv. tomato (Pto) DC3000, the growth of the bacterium was significantly reduced by 2.0- to 2.5-fold compared to that in WT plants. However, the edited alleles enhanced susceptibility against Fusarium oxysporum f. sp. lycopersici, which causes fusarium wilt. CRISPR-Cas9-based precise domain editing of the SlHyPRP1 gene generated multi-stress-tolerant alleles that could be used as genetic materials for tomato breeding.

Improved Dual Base Editor Systems (iACBEs) for Simultaneous Conversion of Adenine and Cytosine in the Bacterium Escherichia coli.

Genome-editing (GE) techniques like base editing are ideal for introducing novel gain-of-function mutations and in situ protein evolution. Features of base editors (BEs) such as higher efficacy, relaxed protospacer adjacent motif (PAM), and a broader editing window enables diversification of user-defined targeted locus. Cytosine (CBE) or adenine (ABE) BEs alone can only alter C-to-T or A-to-G in target sites. In contrast, dual BEs (ACBEs) can concurrently generate C-to-T and A-to-G modifications. Although BE tools have recently been applied in microbes, there is no report of ACBE for microbial GE. In this study, we engineered four improved ACBEs (iACBEs) tethering highly active CBE and ABE variants that can introduce synchronized C-to-T and A-to-G mutations in targeted loci. iACBE4 generated by evoCDA1-ABE9e fusion demonstrated a broader editing window (positions -6 to 15) and is also compatible with the multiplex editing approach in Escherichia coli. We further show that the iACBE4-NG containing PAM-relaxed nCas9-NG expands the targeting scope beyond NGG (N-A/G/C/T) PAM. As a proof-of-concept, iACBE was effectively utilized to identify previously unknown mutations in the rpoB gene, conferring gain-of-function, i.e., rifampicin resistance. The iACBE tool would expand the CRISPR-GE toolkit for microbial genome engineering and synthetic biology. IMPORTANCE Dual base editors are DSB-free CRISPR tools applied in eukaryotes but not yet in bacteria. We developed an improved ACBE toolset for bacteria, combining highly processive deaminases. We believe that the bacterial optimized iACBE toolset is a significant advancement in CRISPR-based E. coli genome editing and adaptable to other microbes.

Engineering drought and salinity tolerance traits in crops through CRISPR-mediated genome editing: Targets, tools, challenges, and perspectives.

Prolonged periods of drought triggered by climate change hamper plant growth and cause substantial agricultural yield losses every year. In addition to drought, salinity is one of the major abiotic stresses that severely affect crop health and agricultural production. Plant responses to drought and salinity involve multiple processes that operate in a spatiotemporal manner, such as stress sensing, perception, epigenetic modifications, transcription, post-transcriptional processing, translation, and post-translational changes. Consequently, drought and salinity stress tolerance are polygenic traits influenced by genome-environment interactions. One of the ideal solutions to these challenges is the development of high-yielding crop varieties with enhanced stress tolerance, together with improved agricultural practices. Recently, genome-editing technologies, especially clustered regularly interspaced short palindromic repeats (CRISPR) tools, have been effectively applied to elucidate how plants deal with drought and saline environments. In this work, we aim to portray that the combined use of CRISPR-based genome engineering tools and modern genomic-assisted breeding approaches are gaining momentum in identifying genetic determinants of complex traits for crop improvement. This review provides a synopsis of plant responses to drought and salinity stresses at the morphological, physiological, and molecular levels. We also highlight recent advances in CRISPR-based tools and their use in understanding the multi-level nature of plant adaptations to drought and salinity stress. Integrating CRISPR tools with modern breeding approaches is ideal for identifying genetic factors that regulate plant stress-response pathways and for the introgression of beneficial traits to develop stress-resilient crops.

Identification and structural analysis of novel malathion-specific DNA aptameric sensors designed for food testing.

Malathion is an organophosphate chemical (OPC) and a toxic contaminant that adversely impacts food quality, human health, biodiversity, and the environment. Due to its small size and unavailability of sensitive sensors, detection of malathion remains a challenging task. Often chromatographic methods employed to analyze OPCs suffer from several shortcomings, including cost, immobility, laboriousness, and unsuitability for point-of-care settings. Hence, developing a specific and sensitive diagnostic sensor for quick and inexpensive food testing is essential. We discovered four unique malathion-specific ssDNA aptamers; designed two independent sensing strategies using fluorescence labeling and Thioflavin T (ThT) displacement. Selected aptamers formed the G4-quadruplex-like (G4Q) structure, which helped develop a label-free detection approach with a 2.01 ppb limit of detection. Additionally, 3D structures of aptamers were generated and validated using a series of computational modeling programs. Furthermore, we explored structural features using CD spectroscopy and molecular docking, probing ligands' binding mode, and revealed vital intermolecular interactions with aptamers. Subsequently, the novel sensors were optimized to detect malathion from food samples. The novel sensors could be further developed to meet the demands of sensing and quantifying toxic contaminants from real food samples in field conditions.

Genome editing and beyond: what does it mean for the future of plant breeding?

MAIN CONCLUSION: Genome editing offers revolutionized solutions for plant breeding to sustain food production to feed the world by 2050. Therefore, genome-edited products are increasingly recognized via more relaxed legislation and community adoption. The world population and food production are disproportionally growing in a manner that would have never matched each other under the current agricultural practices. The emerging crisis is more evident with the subtle changes in climate and the running-off of natural genetic resources that could be easily used in breeding in conventional ways. Under these circumstances, affordable CRISPR-Cas-based gene-editing technologies have brought hope and charged the old plant breeding machine with the most energetic and powerful fuel to address the challenges involved in feeding the world. What makes CRISPR-Cas the most powerful gene-editing technology? What are the differences between it and the other genetic engineering/breeding techniques? Would its products be labeled as "conventional" or "GMO"? There are so many questions to be answered, or that cannot be answered within the limitations of our current understanding. Therefore, we would like to discuss and answer some of the mentioned questions regarding recent progress in technology development. We hope this review will offer another view on the role of CRISPR-Cas technology in future of plant breeding for food production and beyond.

In Vivo Rapid Investigation of CRISPR-Based Base Editing Components in Escherichia coli (IRI-CCE): A Platform for Evaluating Base Editing Tools and Their Components.

Rapid assessment of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-based genome editing (GE) tools and their components is a critical aspect for successful GE applications in different organisms. In many bacteria, double-strand breaks (DSBs) generated by CRISPR/Cas tool generally cause cell death due to the lack of an efficient nonhomologous end-joining pathway and restricts its use. CRISPR-based DSB-free base editors (BEs) have been applied for precise nucleotide (nt) editing in bacteria, which does not need to make DSBs. However, optimization of newer BE tools in bacteria is challenging owing to the toxic effects of BE reagents expressed using strong promoters. Improved variants of two main BEs, cytidine base editor (CBE) and adenine base editor (ABE), capable of converting C to T and A to G, respectively, have been recently developed but yet to be tested for editing characteristics in bacteria. Here, we report a platform for in vivo rapid investigation of CRISPR-BE components in Escherichia coli (IRI-CCE) comprising a combination of promoters and terminators enabling the expression of nCas9-based BE and sgRNA to nontoxic levels, eventually leading to successful base editing. We demonstrate the use of IRI-CCE to characterize different variants of CBEs (PmCDA1, evoCDA1, APOBEC3A) and ABEs (ABE8e, ABE9e) for bacteria, exhibiting that each independent BE has its specific editing pattern for a given target site depending on protospacer length. In summary, CRISPR-BE components expressed without lethal effects on cell survival in the IRI-CCE allow an analysis of various BE tools, including cloned biopart modules and sgRNAs.

The Obstacles and Potential Solution Clues of Prime Editing Applications in Tomato.

Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a "priming" extended guide RNA (gRNA) has shown a high frequency for precise genome modification in mammalian cells and several plant species. Nevertheless, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. Our data obtained by targeted deep sequencing also revealed only low PE efficiencies in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered that the fusion of RT to Cas9 and the structure of PE gRNAs (pegRNAs) negatively affected the cleaving activity of the Cas9 nuclease. The self-complementarity between the primer binding sequences (PBSs) and spacer sequence might pose risks to the activity of the Cas9 complex. However, modifying the pegRNA sequences by shortening or introducing mismatches to the PBSs to reduce their melting temperatures did not enhance the PE efficiency at the MADS-box protein (SlMBP21), alcobaca (SlALC), and acetolactate synthase 1 (SlALS1) loci. Our data show challenges of the PE approach in tomato, indicating that a further improvement of the PE system for successful applications is demanded, such as the use of improved expression systems for enriching active PE complexes.

Pathogen effectors: What do they do at plasmodesmata?

Plants perceive an assortment of external cues during their life cycle, including abiotic and biotic stressors. Biotic stress from a variety of pathogens, including viruses, oomycetes, fungi, and bacteria, is considered to be a substantial factor hindering plant growth and development. To hijack the host cell's defence machinery, plant pathogens have evolved sophisticated attack strategies mediated by numerous effector proteins. Several studies have indicated that plasmodesmata (PD), symplasmic pores that facilitate cell-to-cell communication between a cell and neighbouring cells, are one of the targets of pathogen effectors. However, in contrast to plant-pathogenic viruses, reports of fungal- and bacterial-encoded effectors that localize to and exploit PD are limited. Surprisingly, a recent study of PD-associated bacterial effectors has shown that a number of bacterial effectors undergo cell-to-cell movement via PD. Here we summarize and highlight recent advances in the study of PD-associated fungal/oomycete/bacterial effectors. We also discuss how pathogen effectors interfere with host defence mechanisms in the context of PD regulation.

Single-strand annealing: Molecular mechanisms and potential applications in CRISPR-Cas-based precision genome editing.

BACKGROUND: Spontaneous double-stranded DNA breaks (DSBs) frequently occur within the genome of all living organisms and must be well repaired for survival. Recently, more important roles of the DSB repair pathways that were previously thought to be minor pathways, such as single-strand annealing (SSA), have been shown. Nevertheless, the biochemical mechanisms and applications of the SSA pathway in genome editing have not been updated. PURPOSE AND SCOPE: Understanding the molecular mechanism of SSA is important to design potential applications in gene editing. This review provides insights into the recent progress of SSA studies and establishes a model for their potential applications in precision genome editing. SUMMARY AND CONCLUSION: The SSA mechanism involved in DNA DSB repair appears to be activated by a complex signaling cascade starting with broken end sensing and 5'-3' resection to reveal homologous repeats on the 3' ssDNA overhangs that flank the DSB. Annealing the repeats would help to amend the discontinuous ends and restore the intact genome, resulting in the missing of one repeat and the intervening sequence between the repeats. We proposed a model for CRISPR-Cas-based precision insertion or replacement of DNA fragments to take advantage of the characteristics. The proposed model can add a tool to extend the choice for precision gene editing. Nevertheless, the model needs to be experimentally validated and optimized with SSA-favorable conditions for practical applications.

ROS-mediated plasmodesmal regulation requires a network of an Arabidopsis receptor-like kinase, calmodulin-like proteins, and callose synthases.

Plasmodesmata (PD) play a critical role in symplasmic communication, coordinating plant activities related to growth & development, and environmental stress responses. Most developmental and environmental stress signals induce reactive oxygen species (ROS)-mediated signaling in the apoplast that causes PD closure by callose deposition. Although the apoplastic ROS signals are primarily perceived at the plasma membrane (PM) by receptor-like kinases (RLKs), such components involved in PD regulation are not yet known. Here, we show that an Arabidopsis NOVEL CYS-RICH RECEPTOR KINASE (NCRK), a PD-localized protein, is required for plasmodesmal callose deposition in response to ROS stress. We identified the involvement of NCRK in callose accumulation at PD channels in either basal level or ROS-dependent manner. Loss-of-function mutant (ncrk) of NCRK induces impaired callose accumulation at the PD under the ROS stress resembling a phenotype of the PD-regulating GLUCAN SYNTHASE-LIKE 4 (gsl4) knock-out plant. The overexpression of transgenic NCRK can complement the callose and the PD permeability phenotypes of ncrk mutants but not kinase-inactive NCRK variants or Cys-mutant NCRK, in which Cys residues were mutated in Cys-rich repeat ectodomain. Interestingly, NCRK mediates plasmodesmal permeability in mechanical injury-mediated signaling pathways regulated by GSL4. Furthermore, we show that NCRK interacts with calmodulin-like protein 41 (CML41) and GSL4 in response to ROS stress. Altogether, our data indicate that NCRK functions as an upstream regulator of PD callose accumulation in response to ROS-mediated stress signaling pathways.

Improvement of the LbCas12a-crRNA System for Efficient Gene Targeting in Tomato.

Plant gene targeting (GT) can be utilized to precisely replace up to several kilobases of a plant genome. Recent studies using the powerful clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nucleases significantly improved plant GT efficiency. However, GT for loci without associated selection markers is still inefficient. We previously utilized Lachnospiraceae bacterium Cas12a (LbCas12a) in combination with a replicon for tomato GT and obtained high GT efficiency with some selection markers. In this study, we advance our GT system by inhibiting the cNHEJ pathway with small chemical molecules such as NU7441. Further optimization of the GT is also possible with the treatment of silver nitrate possibly via its pronounced actions in ethylene inhibition and polyamine production. Importantly, the GT efficiency is significantly enhanced with the use of a temperature-tolerant LbCas12a (ttLbCas12a) that is capable of performing target cleavage even at low temperatures. Targeted deep sequencing, as well as conventional methods, are used for the assessment of the editing efficiency at both cell and plant levels. Our work demonstrates the significance of the selection of gene scissors, the appropriate design and number of LbCas12a crRNAs, the use of chemical treatments, and the establishment of favorable experimental conditions for further enhancement of plant HDR to enable efficient GT in tomato.

Salinity Stress in Potato: Understanding Physiological, Biochemical and Molecular Responses.

Among abiotic stresses, salinity is a major global threat to agriculture, causing severe damage to crop production and productivity. Potato (Solanum tuberosum) is regarded as a future food crop by FAO to ensure food security, which is severely affected by salinity. The growth of the potato plant is inhibited under salt stress due to osmotic stress-induced ion toxicity. Salinity-mediated osmotic stress leads to physiological changes in the plant, including nutrient imbalance, impairment in detoxifying reactive oxygen species (ROS), membrane damage, and reduced photosynthetic activities. Several physiological and biochemical phenomena, such as the maintenance of plant water status, transpiration, respiration, water use efficiency, hormonal balance, leaf area, germination, and antioxidants production are adversely affected. The ROS under salinity stress leads to the increased plasma membrane permeability and extravasations of substances, which causes water imbalance and plasmolysis. However, potato plants cope with salinity mediated oxidative stress conditions by enhancing both enzymatic and non-enzymatic antioxidant activities. The osmoprotectants, such as proline, polyols (sorbitol, mannitol, xylitol, lactitol, and maltitol), and quaternary ammonium compound (glycine betaine) are synthesized to overcome the adverse effect of salinity. The salinity response and tolerance include complex and multifaceted mechanisms that are controlled by multiple proteins and their interactions. This review aims to redraw the attention of researchers to explore the current physiological, biochemical and molecular responses and subsequently develop potential mitigation strategies against salt stress in potatoes.