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Tobacco (Nicotiana tabacum) is known for its psychoactive alkaloid nicotine, which presents significant public health challenges. Recent research has linked the final stages of nicotine biosynthesis with the BBL and A622 genes, yet this part of the biosynthetic pathway remains largely unexplored, representing a 'black box' in our understanding. In our study, we employed a multi-target CRISPR/Cas9 system to target homologous genes of BBL and A622 in commercial tobacco varieties Virginia, creating various mutants. This led to significant variations in plant development and alkaloid content. Notably, mutant lines a622a-38–5 and a622l-3–9 with exon-intron boundary deletions exhibited significantly decreased plant height and leaf number, along with a substantial reduction in alkaloids, including nicotine. Particularly, double mutants in the A622 family displayed more severe effects than sextuple BBL mutants, emphasizing the distinctive role of A622 in nicotine synthesis and plant development. Our findings demonstrate that mutations in A622 and BBL genes can drastically reduce nicotine and anatabine content, with some cases showing reductions up to 99.6%. These results underscore the potential of genome editing in developing tobacco varieties with significantly lower nicotine levels. This study not only enhances our understanding of nicotine biosynthesis but also contributes to public health efforts by providing a pathway to develop less addictive tobacco products.

Climate change presents numerous challenges for agriculture, including frequent events of plant abiotic stresses such as elevated temperatures that lead to heat stress (HS). As the primary driving factor of climate change, HS threatens global food security and biodiversity. In recent years, HS events have negatively impacted plant physiology, reducing plant’s ability to maintain disease resistance and resulting in lower crop yields. Plants must adapt their priorities toward defense mechanisms to tolerate stress in challenging environments. Furthermore, selective breeding and long-term domestication for higher yields have made crop varieties vulnerable to multiple stressors, making them more susceptible to frequent HS events. Studies on climate change predict that concurrent HS and biotic stresses will become more frequent and severe in the future, potentially occurring simultaneously or sequentially. While most studies have focused on singular stress effects on plant systems to examine how plants respond to specific stresses, the simultaneous occurrence of HS and biotic stresses pose a growing threat to agricultural productivity. Few studies have explored the interactions between HS and plant–biotic interactions. Here, we aim to shed light on the physiological and molecular effects of HS and biotic factor interactions (bacteria, fungi, oomycetes, nematodes, insect pests, pollinators, weedy species, and parasitic plants), as well as their combined impact on crop growth and yields. We also examine recent advances in designing and developing various strategies to address multi-stress scenarios related to HS and biotic factors.

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.

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.

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.

ABSTRACT 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.

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.

Halim Park and Jin Hwa Park et al. report the nuclear genome sequence of the duckweed Wolffia australiana, the smallest known flowering plant. The genome assembly represents an improvement over a recently published genome and highlights genome rearrangements that may be linked to its specialized aquatic adaptations.

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.

Abstract Continuing crop domestication/redomestication and modification is a key determinant of the adaptation and fulfillment of the food requirements of an exploding global population under increasingly challenging conditions such as climate change and the reduction in arable lands. Monocotyledonous crops are not only responsible for approximately 70% of total global crop production, indicating their important roles in human life, but also the first crops to be challenged with the abovementioned hurdles; hence, monocot crops should be the first to be engineered and/or de novo domesticated/redomesticated. A long time has passed since the first green revolution; the world is again facing the challenge of feeding a predicted 9.7 billion people in 2050, since the decline in world hunger was reversed in 2015. One of the major lessons learned from the first green revolution is the importance of novel and advanced trait-carrying crop varieties that are ideally adapted to new agricultural practices. New plant breeding techniques (NPBTs), such as genome editing, could help us succeed in this mission to create novel and advanced crops. Considering the importance of NPBTs in crop genetic improvement, we attempt to summarize and discuss the latest progress with major approaches, such as site-directed mutagenesis using molecular scissors, base editors and especially homology-directed gene targeting (HGT), a very challenging but potentially highly precise genome modification approach in plants. We therefore suggest potential approaches for the improvement of practical HGT, focusing on monocots, and discuss a potential approach for the regulation of genome-edited products.

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.

Plasmodesmata (PD) are cytoplasmic canals that facilitate intercellular communication and molecular exchange between adjacent plant cells. PD-associated proteins are considered as one of the foremost factors in regulating PD function that is critical for plant development and stress responses. Although its potential to be used for crop engineering is enormous, our understanding of PD biology was relatively limited to model plants, demanding further studies in crop systems. Recently developed genome editing techniques such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associate protein (CRISPR/Cas) might confer powerful approaches to dissect the molecular function of PD components and to engineer elite crops. Here, we assess several aspects of PD functioning to underline and highlight the potential applications of CRISPR/Cas that provide new insight into PD biology and crop improvement.

Currently, poor biodiversity has raised challenges in the breeding and cultivation of tomatoes, which originated from the Andean region of Central America, under global climate change. Meanwhile, the wild relatives of cultivated tomatoes possess a rich source of genetic diversity but have not been extensively used for the genetic improvement of cultivated tomatoes due to the possible linkage drag of unwanted traits from their genetic backgrounds. With the advent of new plant breeding techniques (NPBTs), especially CRISPR/Cas-based genome engineering tools, the high-precision molecular breeding of tomato has become possible. Further, accelerated introgression or de novo domestication of novel and elite traits from/to the wild tomato relatives to/from the cultivated tomatoes, respectively, has emerged and has been enhanced with high-precision tools. In this review, we summarize recent progress in tomato precision genome editing and its applications for breeding, with a special focus on CRISPR/Cas-based approaches. Future insights and precision tomato breeding scenarios in the CRISPR/Cas era are also discussed.

Lateral root development is known to be regulated by Aux/IAA-ARF modules in Arabidopsisthaliana. As components, several Aux/IAAs have participated in these Aux/IAA-ARF modules. In this study, to identify the biological function of IAA15 in plant developments, transgenic plant overexpressing the gain-of-function mutant of IAA15 (IAA15P75S OX) under the control of dexamethasone (DEX) inducible promoter, in which IAA15 protein was mutated by changing Pro-75 residue to Ser at the degron motif in conserved domain II, was constructed. As a result, we found that IAA15P75S OX plants show a decreased number of lateral roots. Coincidently, IAA15 promoter-GUS reporter analysis revealed that IAA15 transcripts were highly detected in all stages of developing lateral root tissues. It was also verified that the IAA15P75S protein is strongly stabilized against proteasome-mediated protein degradation by inhibiting its poly-ubiquitination, resulting in the transcriptional repression of auxin-responsive genes. In particular, transcript levels of LBD16 and LBD29, which are positive regulators of lateral root formation, dramatically repressed in IAA15P75S OX plants. Furthermore, it was elucidated that IAA15 interacts with ARF7 and ARF19 and binds to the promoters of LBD16 and LBD29, strongly suggesting that IAA15 represses lateral root formation through the transcriptional suppression of LBD16 and LBD29 by inhibiting ARF7 and ARF19 activity. Taken together, this study suggests that IAA15 also plays a key negative role in lateral root formation as a component of Aux/IAA-ARF modules.

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.

Cell surface receptors perceive signals from the environment and transfer them to the interior of the cell. The Arabidopsis thaliana PR5 receptor-like kinase (AtPR5K) subfamily consists of three members with extracellular domains that share sequence similarity with the PR5 proteins. In this study, we characterized the role of AtPR5K2 in plant drought-stress signaling. AtPR5K2 is predominantly expressed in leaves and localized to the plasma membrane. The atpr5k2-1 mutant showed tolerance to dehydration stress, while AtPR5K2-overexpressing plants was hypersensitive to drought. Bimolecular fluorescence complementation assays showed that AtPR5K2 physically interacted with the type 2C protein phosphatases ABA-insensitive 1 (ABI1) and ABI2 and the SNF1-related protein kinase 2 (SnRK2.6) proteins, all of which are involved in the initiation of abscisic acid (ABA) signaling; however, these interactions were inhibited by treatments of exogenous ABA. Moreover, AtPR5K2 was found to phosphorylate ABI1 and ABI2, but not SnRK2.6. Taken together, these results suggest that AtPR5K2 participates in ABA-dependent drought-stress signaling through the phosphorylation of ABI1 and ABI2.

Over the last decade, plasmodesmata (PD) symplasmic nano-channels were reported to be involved in various cell biology activities to prop up within plant growth and development as well as environmental stresses. Indeed, this is highly influenced by their native structure, which is lined with the plasma membrane (PM), conferring a suitable biological landscape for numerous plant receptors that correspond to signaling pathways. However, there are more than six hundred members of Arabidopsis thaliana membrane-localized receptors and over one thousand receptors in rice have been identified, many of which are likely to respond to the external stimuli. This review focuses on the class of plasmodesmal-receptor like proteins (PD-RLPs)/plasmodesmal-receptor-like kinases (PD-RLKs) found in planta. We summarize and discuss the current knowledge regarding RLPs/RLKs that reside at PD−PM channels in response to plant growth, development, and stress adaptation.

Rubus coreanus Miquel is a Korean black raspberry used in folk medicine and functional foods. To investigate the biosynthesis pathway of anthocyanin in R. coreanus Miquel, the complete coding sequence of flavonoid 3′-hydroxylase (F3′H), designated as RcMF3′H1, was cloned for the first time using the Korean black raspberry transcriptome library. The deduced amino acid sequence of RcMF3′H1 contained the proline-rich “hinge” region, P450 consensus heme-binding domain, and F3′H-specific motifs. Phylogenetic analysis revealed that RcMF3′H1 was clustered into the same subgroup as other plant F3′Hs. In addition, expression analysis by quantitative real-time PCR revealed the involvement of RcMF3′H1 in methyl jasmonate-mediated anthocyanin biosynthesis. Furthermore, the ability of the RcMF3′H1 gene to complement the Arabidopsis transparent testa 7-1 mutant suggested that RcMF3′H1 encodes the functional F3′H enzyme involved in anthocyanin biosynthesis. Taken together, the cloning and molecular characterization of RcMF3′H1 will facilitate a better insight into the anthocyanin biosynthesis pathway in R. coreanus Miquel.

Plants and microbes are co-evolved and interact with each other in nature. Plant-associated microbes, often referred to as plant microbiota, are an integral part of plant life. Depending on the health effects on hosts, plant−microbe (PM) interactions are either beneficial or harmful. The role of microbiota in plant growth promotion (PGP) and protection against various stresses is well known. Recently, our knowledge of community composition of plant microbiome and significant driving factors have significantly improved. So, the use of plant microbiome is a reliable approach for a next green revolution and to meet the global food demand in sustainable and eco-friendly agriculture. An application of the multifaceted PM interactions needs the use of novel tools to know critical genetic and molecular aspects. Recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-mediated genome editing (GE) tools are of great interest to explore PM interactions. A systematic understanding of the PM interactions will enable the application of GE tools to enhance the capacity of microbes or plants for agronomic trait improvement. This review focuses on applying GE techniques in plants or associated microbiota for discovering the fundamentals of the PM interactions, disease resistance, PGP activity, and future implications in agriculture.

Communication between cells is an essential process for developing and maintaining multicellular collaboration during plant development and physiological adaptation in response to environmental stimuli. The intercellular movement of proteins and RNAs in addition to the movement of small nutrients or signaling molecules such as sugars and phytohormones has emerged as a novel mechanism of cell-to-cell signaling in plants. As a strategy for efficient intercellular communication and long-distance molecule movement, plants have evolved plant-specific symplasmic communication networks via plasmodesmata (PDs) and the phloem.

Abstract: The specialized plasma membrane microdomains known as lipid rafts are enriched by sterols and sphingolipids. Lipid rafts facilitate cellular signal transduction by controlling the assembly of signaling molecules and membrane protein trafficking. Another specialized compartment of plant cells, the plasmodesmata (PD), which regulates the symplasmic intercellular movement of certain molecules between adjacent cells, also contains a phospholipid bilayer membrane. The dynamic permeability of plasmodesmata (PDs) is highly controlled by plasmodesmata callose (PDC), which is synthesized by callose synthases (CalS) and degraded by β-1,3-glucanases (BGs). In recent studies, remarkable observations regarding the correlation between lipid raft formation and symplasmic intracellular trafficking have been reported, and the PDC has been suggested to be the regulator of the size exclusion limit of PDs. It has been suggested that the alteration of lipid raft substances impairs PDC homeostasis, subsequently affecting PD functions. In this review, we discuss the substantial role of membrane lipid rafts in PDC homeostasis and provide avenues for understanding the fundamental behavior of the lipid raft–processed PDC.

The Korean black raspberry (Rubus coreanus Miquel, KB) on ripening is usually consumed as fresh fruit, whereas the unripe KB has been widely used as a source of traditional herbal medicine. Such a stage specific utilization of KB has been assumed due to the changing metabolite profile during fruit ripening process, but so far molecular and biochemical changes during its fruit maturation are poorly understood. To analyze biochemical changes during fruit ripening process at molecular level, firstly, we have sequenced, assembled, and annotated the transcriptome of KB fruits. Over 4.86 Gb of normalized cDNA prepared from fruits was sequenced using Illumina HiSeq™ 2000, and assembled into 43,723 unigenes. Secondly, we have reported that alterations in anthocyanins and proanthocyanidins are the major factors facilitating variations in these stages of fruits. In addition, up-regulation of F3'H1, DFR4 and LDOX1 resulted in the accumulation of cyanidin derivatives during the ripening process of KB, indicating the positive relationship between the expression of anthocyanin biosynthetic genes and the anthocyanin accumulation. Furthermore, the ability of RcMCHI2 (R. coreanus Miquel chalcone flavanone isomerase 2) gene to complement Arabidopsis transparent testa 5 mutant supported the feasibility of our transcriptome library to provide the gene resources for improving plant nutrition and pigmentation. Taken together, these datasets obtained from transcriptome library and metabolic profiling would be helpful to define the gene-metabolite relationships in this non-model plant.