Oligomeric amyloid-ß targeted contrast agent for MRI evaluation of Alzheimer's disease mouse models.

Background: Oligomeric amyloid beta (oAß) is a toxic factor that acts in the early stage of Alzheimer's disease (AD) and may initiate the pathologic cascade. Therefore, detecting oAß has a crucial role in the early diagnosis, monitoring, and treatment of AD. Purpose: The purpose of this study was to evaluate MRI signal changes in different mouse models and the time-dependent signal changes using our novel gadolinium (Gd)-dodecane tetraacetic acid (DOTA)- ob5 aptamer contrast agent. Methods: We developed an MRI contrast agent by conjugating Gd-DOTA-DNA aptamer called ob5 to evaluate its ability to detect oAß deposits in the brain using MRI. A total of 10 control mice, 9 3xTg AD mice, and 11 APP/PS/Tau AD mice were included in this study, with the age of each model being 16 or 36 weeks. A T1-weighted image was acquired at the time points before (0 min) and after injection of the contrast agent at 5, 10, 15, 20, and 25 min. The analyses were performed to compare MRI signal differences among the three groups and the time-dependent signal differences in different mouse models. Results: Both 3xTg AD and APP/PS/Tau AD mouse models had higher signal enhancement than control mice at all scan-time points after injection of our contrast media, especially in bilateral hippocampal areas. In particular, all Tg AD mouse models aged 16 weeks showed a higher contrast enhancement than those aged 36 weeks. For 3xTg AD and APP/PS/Tau AD groups, the signal enhancement was significantly different among the five time points (0 min, 5 min, 10 min, 15 min, 20 min, and 25 min) in multiple ROI areas, typically in the bilateral hippocampus, left thalamus, and left amygdala. Conclusion: The findings of this study suggest that the expression of the contrast agent in different AD models demonstrates its translational flexibility across different species. The signal enhancement peaked around 15-20 min after injection of the contrast agent. Therefore, our novel contrast agent targeting oAß has the potential ability to diagnose early AD and monitor the progression of AD.

Comparison of the Pharmacokinetics of Gadolinium-Based and Iron Oxide-Based Contrast Agents inside the Lymphatic Structure using Magnetic Resonance Lymphangiography.

PURPOSE: Gadolinium (Gd)-based contrast agents are primarily used for contrast-enhanced magnetic resonance lymphangiography (MRL). However, overcoming venous contamination issues remains challenging. This study aims to assess the MRL efficacy of the newly developed iron-based contrast agent (INV-001) that is specially designed to mitigate venous contamination issues. The study further explores the optimal dosage, including both injection volume and concentration, required to achieve successful visualization of the popliteal lymph nodes and surrounding lymphatic vessels. PROCEDURES: All animals utilized in this study were male Sprague-Dawley (SD) rats weighing between 250 and 300 g. The contrast agents prepared were injected intradermally in the fourth phalanx of both hind limbs using a 30-gauge syringe in SD rats. MRL was performed every 16 min on a coronal 3D time-of-flight sequence with saturation bands using a 9.4-T animal machine. RESULTS: Contrary to Gd-DOTA, which exhibited venous contamination in most animals irrespective of injection dosages and conditions, INV-001 showed no venous contamination. For Gd-DOTA, the popliteal lymph nodes and lymphatic vessels reached peak enhancement 16 min after injection from the injection site and then rapidly washed out. However, with INV-001, they reached peak enhancement between 16 and 32 min after injection, with prolonged visualization of the popliteal lymph node and lymphatic vessels. INV-001 at 0.45 µmol (15 mM, 30 µL) and 0.75 µmol (15 mM, 50 µL) achieved high scores for qualitative image analysis, providing good visualization of the popliteal lymph nodes and lymphatic vessels without issues of venous contamination, interstitial space enhancement, or lymph node enlargement. CONCLUSION: In MRL, INV-001, a novel T1 contrast agent based on iron, enables prolonged enhancement of popliteal lymph nodes and lymphatic vessels without venous contamination.

Margin of exposure to free formaldehyde in personal care products containing formaldehyde-donor preservatives: Evidence for consumer safety.

Formaldehyde has been classified as carcinogenic to humans by International Agency for Research on Cancer and found in personal care (PC) products containing formaldehyde-donor (FD) preservatives. However, the cancer risk associated with the use of FD-containing PC products has not been well established. Our study provides the quantitative cancer risk assessment of formaldehyde in FD-containing PC products. The carbon-13 nuclear magnetic resonance (13C-NMR) spectroscopy was used in this risk assessment to provide reliable exposure information to formaldehyde in PC products and aqueous solutions containing sodium hydroxymethylglycinate. The risk assessment was conducted using the margin of exposure (MOE) approach with benchmark doses (BMDs) for 10% effect. For hemolymphoreticular neoplasias in male rats, a BMD of 28.03 mg/kg/day and a BMD lower confidence limit (BMDL) of 2.52 mg/kg/day were calculated from available long-term animal experiments. The worst-case consumer exposure to formaldehyde from FD-containing PC products was 0.007 µg/kg/day. Comparing the consumer exposure with BMDL, the resulting MOE was 360,000 for the worst-case scenario. The consumer exposure to formaldehyde (0.007 µg/kg/day) from using FD-containing PC products represents less than 1.0 × 10-6 % of background level endogenous formaldehyde (878-1310 mg/kg/day). The cancer risk from formaldehyde to consumers using FD-containing PC products is negligible.

Enhancement of osmotic stress tolerance in soybean seed germination by bacterial bioactive extracts.

Soybean (Glycine max (L.) Merr.) is important to the global food industry; however, its productivity is affected by abiotic stresses such as osmosis, flooding, heat, and cold. Here, we evaluated the bioactive extracts of two biostimulant bacterial strains, Bacillus butanolivorans KJ40 and B. siamensis H30-3, for their ability to convey tolerance to osmotic stress in soybean seeds during germination. Soybean seeds were dip-treated in extracts of KJ40 (KJ40E) or H30-3 (H30-3E) and incubated with either 0% or 20% polyethylene glycol 6000 (PEG), simulating drought-induced osmotic stress. We measured malondialdehyde content as a marker for lipid peroxidation, as well as the activity of antioxidant enzymes, including catalase, glutathione peroxidase, and glutathione reductase, together with changes in sugars content. We also monitored the expression of genes involved in the gibberellic acid (GA)-biosynthesis pathway, and abscisic acid (ABA) signaling. Following osmotic stress in the extract-treated seeds, malondialdehyde content decreased, while antioxidant enzyme activity increased. Similarly, the expression of GA-synthesis genes, including GmGA2ox1 and GmGA3 were upregulated in KJ40E-dipped seeds at 12 or 6 h after treatment, respectively. The ABA signaling genes GmABI4 and GmDREB1 were upregulated in H30-3E- and KJ40E-treated seeds at 0 and 12 h after treatment under osmotic stress; however, GmABI5, GmABI4, and GmDREB1 levels were also elevated in the dip-treated seeds in baseline conditions. The GA/ABA ratio increased only in KJ40E-treated seeds undergoing osmotic stress, while glucose content significantly decreased in H30-3E-treated seeds at 24 h after treatment. Collectively, our findings indicated that dip-treatment of soybean seeds in KJ40E and H30-3E can enhance the seeds' resistance to osmotic stress during germination, and ameliorate cellular damage caused by secondary oxidative stress. This seed treatment can be used agriculturally to promote germination under drought stress and lead to increase crop yield and quality.

Is aryl hydrocarbon receptor antagonism after ischemia effective in alleviating acute hepatic ischemia-reperfusion injury in rats?

Aryl hydrocarbon receptors (AhRs) have been reported to be important mediators of ischemic injury in the brain. Furthermore, the pharmacological inhibition of AhR activation after ischemia has been shown to attenuate cerebral ischemia-reperfusion (IR) injury. Here, we investigated whether AhR antagonist administration after ischemia was also effective in ameliorating hepatic IR injury. A 70% partial hepatic IR (45-min ischemia and 24-h reperfusion) injury was induced in rats. We administered 6,2',4'-trimethoxyflavone (TMF, 5 mg/kg) intraperitoneally 10 min after ischemia. Hepatic IR injury was observed using serum, magnetic resonance imaging-based liver function indices, and liver samples. TMF-treated rats showed significantly lower relative enhancement (RE) values and serum alanine aminotransferase (ALT) and aspartate aminotransferase levels than did untreated rats at 3 h after reperfusion. After 24 h of reperfusion, TMF-treated rats had significantly lower RE values, ΔT1 values, serum ALT levels, and necrotic area percentage than did untreated rats. The expression of the apoptosis-related proteins, Bax and cleaved caspase-3, was significantly lower in TMF-treated rats than in untreated rats. This study demonstrated that inhibition of AhR activation after ischemia was effective in ameliorating IR-induced liver injury in rats.

High-quality genome assembly and genetic mapping reveal a gene regulating flesh color in watermelon (Citrullus lanatus).

The unique color and type characteristics of watermelon fruits are regulated by many molecular mechanisms. However, it still needs to be combined with more abundant genetic data to fine-tune the positioning. We assembled genomes of two Korean inbred watermelon lines (cv. 242-1 and 159-1) with unique color and fruit-type characteristics and identified 23,921 and 24,451 protein-coding genes in the two genomes, respectively. To obtain more precise results for further study, we resequenced one individual of each parental line and an F2 population composed of 87 individuals. This identified 1,539 single-nucleotide polymorphisms (SNPs) and 80 InDel markers that provided a high-density genetic linkage map with a total length of 3,036.9 cM. Quantitative trait locus mapping identified 15 QTLs for watermelon fruit quality-related traits, including ß-carotene and lycopene content in fruit flesh, fruit shape index, skin thickness, flesh color, and rind color. By investigating the mapping intervals, we identified 33 candidate genes containing variants in the coding sequence. Among them, Cla97C01G008760 was annotated as a phytoene synthase with a single-nucleotide variant (A → G) in the first exon at 9,539,129 bp of chromosome 1 that resulted in the conversion of a lysine to glutamic acid, indicating that this gene might regulate flesh color changes at the protein level. These findings not only prove the importance of a phytoene synthase gene in pigmentation but also explain an important reason for the color change of watermelon flesh.

The absence of genotoxicity of Aloe vera beverages: A review of the literature.

Aloe has a long history of topical and systemic use with testimonials of countless health benefits and is one of the most popular botanical medicines in the world for the management of a wide variety both of benign and serious ailments including irritable bowel syndromes, osteoarthritis, Type II diabetes mellitus, and viral respiratory illness. The human consumption of Aloe vera extract in beverage form has substantially grown over the last several decades, in no small part, due to the increased consumer interest in alternative approaches to health benefits. The principal aim of the present paper is to characterize the research to date that has explored the genotoxic potential of Aloe vera inner leaf gel extract and decolorized whole leaf extract used in commercially available food-grade drinkable products which contain no more than 10 ppm aloin. Despite prevailing public health opinion, especially in Europe, the consensus of the reviewed studies retrieved from the peer-reviewed literature together with a mutagenic evaluation of an Aloe vera whole leaf decolorized spray-dried powder is that these products are not genotoxic.

MicroRNA super-resolution imaging in blood for Alzheimer's disease.

We propose a novel blood biomarker detection method that uses miRNA super-resolution imaging to enable the early diagnosis of Alzheimer's disease (AD). Here, we report a singlemolecule detection method for visualizing disease-specific miRNA in tissue from an AD mice model, and peripheral blood mononuclear cells (PBMCs) from AD patients. Using optimized Magnified Analysis of Proteome (MAPs), we confirmed that five miRNAs contribute to neurodegenerative disease in the brain hippocampi of 5XFAD and wild-type mice. We also assessed PBMCs isolated from the whole blood of AD patients and a healthy control group, and subsequently analyzed those samples using miRNA super-resolution imaging. We detected more miR-200a-3p expression in the cornu ammonis 1 and dentate gyrus regions of 3 month-old 5XFAD mice than in wild-type mice. Additionally, miRNA super-resolution imaging of blood provides AD diagnosis platform for studying miRNA regulation inside cells at the single molecule level. Our results present a potential liquid biopsy method that could improve the diagnosis of early stage AD and other diseases. [BMB Reports 2023; 56(3): 190-195].

An aptamer-based magnetic resonance imaging contrast agent for detecting oligomeric amyloid-ß in the brain of an Alzheimer's disease mouse model.

The oligomeric amyloid-ß (oAß) is a reliable feature for an early diagnosis of Alzheimer's disease (AD). Therefore, the objective of this study was to demonstrate imaging of oAß deposits using our developed DNA aptamer called ob5 conjugated with gadolinium (Gd)-dodecane tetraacetic acid (DOTA) as a contrast agent for early diagnosis of AD using MRI. An oAß-specific aptamer was developed by amide bond formation and conjugated to Gd-DOTA MRI contrast agent and/or cyanine5 (cy5). We verified the performance of our new contrast agent with an AD mouse model using in vivo and ex vivo fluorescent imaging and animal MRI experiments. The presence of soluble Aß in 3xTg AD mice was detected using GdDOTA-ob5-cy5 probe ex vivo. Fluorescence intensities of the GdDOTA-ob5-cy5 contrast agent were high in the brains of 3xTg-AD mice, but relatively low in the brains of control mice. The GdDOTA-ob5 contrast agent had higher relaxivity than a clinically available contrast agent. T1-weighted MRI signals in 5-month-old 3xTg AD mice increased at 5 min, were prolonged until 10 min, then decreased 15 min after injecting the GdDOTA-ob5 contrast agent. Our targeted DNA aptamer GdDOTA-ob5 contrast agent could be potentially useful for validating the efficacy of a novel diagnostic contrast agent for selectively targeting neurotoxic oAß. It could ultimately be used for early diagnosis of AD.

Preclinical Long-term Magnetic Resonance Imaging Study of Silymarin Liver-protective Effects.

Background and Aims: Efficacy evaluations with preclinical magnetic resonance imaging (MRI) are uncommon, but MRI in the preclinical phase of drug development provides information that is useful for longitudinal monitoring. The study aim was to monitor the protective effectiveness of silymarin with multiparameter MRI and biomarkers in a thioacetamide (TAA)-induced model of liver injury in rats. Correlation analysis was conducted to assess compare the monitoring of liver function by MRI and biomarkers. Methods: TAA was injected three times a week for 8 weeks to generate a disease model (TAA group). In the TAA and silymarin-treated (TAA-SY) groups, silymarin was administered three times weekly from week 4. MR images were acquired at 0, 2, 4, 6, and 8 weeks in the control, TAA, and TAA-SY groups. Results: The area under the curve to maximum time (AUCtmax) and T2* values of the TAA group decreased over the study period, but the serological markers of liver abnormality increased significantly more than those in the control group. In the TAA-SY group, MRI and serological biomarkers indicated attenuation of liver function as in the TAA group. However, pattern changes were observed from week 6 to comparable levels in the control group with silymarin treatment. Negative correlations between either AUCtmax or T2* values and the serological biomarkers were observed. Conclusions: Silymarin had hepatoprotective effects on TAA-induced liver injury and demonstrated the usefulness of multiparametric MRI to evaluate efficacy in preclinical studies of liver drug development.

Comprehensive Genome-Wide Analysis and Expression Pattern Profiling of the SlHVA22 Gene Family Unravels Their Likely Involvement in the Abiotic Stress Adaptation of Tomato.

HVA22 family proteins with a conserved TB2/DP1/HVA22 domain are ubiquitous in eukaryotes. HVA22 family genes have been identified in a variety of plant species. However, there has been no comprehensive genome-wide analysis of HVA22 family genes in tomato (Solanum lycopersicum L.). Here, we identified 15 non-redundant SlHVA22 genes with three segmentally duplicated gene pairs on 8 of the 12 tomato chromosomes. The predicted three-dimensional (3D) models and gene ontology (GO) annotations of SlHVA22 proteins pointed to their putative transporter activity and ability to bind to diverse ligands. The co-expression of SlHVA22 genes with various genes implicated in multiple metabolic pathways and the localization of SlHVA22-GFP fused proteins to the endoplasmic reticulum suggested that they might have a variety of biological functions, including vesicular transport in stressed cells. Comprehensive expression analysis revealed that SlHVA22 genes were differentially expressed in various organs and in response to abiotic stress conditions. The predominant expression of SlHVA22i at the ripening stage and that of SlHVA22g, SlHVA22k, and SlHVA22l in fruits at most developmental stages suggested their probable involvement in tomato fruit development and ripening. Moreover, the transcript expression of most tomato HVA22 genes, particularly SlHVA22b, SlHVA22i, SlHVA22k, SlHVA22l, SlHVA22m, and SlHVA22n, was affected by abscisic acid (ABA) and diverse abiotic stress treatments, indicating the likely involvement of these genes in tomato abiotic stress responses in an ABA-dependent manner. Overall, our findings provide a foundation to better understand the structures and functional roles of SlHVA22 genes, many of which might be useful to improve the abiotic stress tolerance and fruit quality of tomato through marker-assisted backcrossing or transgenic approaches.

Ultrasensitive probeless capacitive biosensor for amyloid beta (Aß1-42) detection in human plasma using interdigitated electrodes.

Progressive aggregation and protein misfolding are the initial fundamental indicators of neurodegenerative disorders such as Alzheimer's disease (AD). In this study, a highly sensitive and novel method to detect amyloid beta (Aß) biomarkers, which are a hallmark of AD, using an immunoassay platform-based interdigitated capacitive biosensor, has been explored. For several decades, aptamers have classified as a novel class of molecular recognition probes comprising single-stranded complementary DNA sequences that bind to their identified targets with high specificity and affinity by an in vitro technique called SELEX (systematic evolution of exponential and enrichment). Aptamers, often referred to as "chemical antibodies", possess several highly obvious features for clinical use. The proposed sensing bio-device was fabricated and glazed with oligomeric Aß (oAß) aptamer and anti-oAß antibody, functionalized onto a Pt/Ti-featured SiO2 substrate. Subsequently, analytical studies were conducted to confirm that the specificity, sensitivity, and selective detection of the oAß-based bioengineered surfaces facilitate a label-free approach. The bionic capacitive sensor achieved real-time detection within 5 s (faster response than ELISA) under the femto-molar range concentrations of oAß peptide in plasma using anti-oAß antibody and oAß aptamer with ultra-high affinity. Furthermore, the prepared capacitive biochip was selective against plasma-borne antigens and standby for 100 days at 4 °C. The developed biosensor is suitable for point-of-care (POC) diagnostic applications owing to its portability and scalability. Furthermore, the superior efficacy of oAß in identifying AD has huge potential for biomedical applications.

The complete chloroplast genome of Utricularia tenuicaulis Miki (Lentibulariaceae) isolated in Korea.

Utricularia tenuicaulis Miki 1935 is an aquatic carnivorous plant species found in East Asia including Korea and Japan. In this study, the chloroplast genome of U. tenuicaulis was successfully sequenced. The assembled genome (153,976 bp; GC ratio, 37.0%) contains four subregions, with the large single copy (LSC; 84,596 bp; 34.9%) and small single copy (SSC; 17,946 bp; 30.5%) regions separated by 25,718 bp of inverted repeat regions (42.7%), and includes 126 genes (81 protein-coding genes, 8 rRNAs, and 37 tRNAs). Phylogenetic analyses based on the whole-chloroplast genomes of 18 species, including 17 Lentibulariaceae species and one outgroup species, suggest a close relationship between U. tenuicaulis and Utricularia macrorhiza Leconte 1824. A comparison of genomic variation between U. tenuicaulis and U. macrorhiza confirmed the validity of the specific discrimination of U. tenuicaulis.

Genotyping-by-sequencing-based identification of Arabidopsis pattern recognition receptor RLP32 recognizing proteobacterial translation initiation factor IF1.

Activation of plant pattern-triggered immunity (PTI) relies on the recognition of microbe-derived structures, termed patterns, through plant-encoded surface-resident pattern recognition receptors (PRRs). We show that proteobacterial translation initiation factor 1 (IF1) triggers PTI in Arabidopsis thaliana and related Brassicaceae species. Unlike for most other immunogenic patterns, IF1 elicitor activity cannot be assigned to a small peptide epitope, suggesting that tertiary fold features are required for IF1 receptor activation. We have deployed natural variation in IF1 sensitivity to identify Arabidopsis leucine-rich repeat (LRR) receptor-like protein 32 (RLP32) as IF1 receptor using a restriction site-associated DNA sequencing approach. RLP32 confers IF1 sensitivity to rlp32 mutants, IF1-insensitive Arabidopsis accessions and IF1-insensitive Nicotiana benthamiana, binds IF1 specifically and forms complexes with LRR receptor kinases SOBIR1 and BAK1 to mediate signaling. Similar to other PRRs, RLP32 confers resistance to Pseudomonas syringae, highlighting an unexpectedly complex array of bacterial pattern sensors within a single plant species.

The complete chloroplast genome of Atriplex gmelinii C. A. Mey. ex Bong. (Amaranthaceae).

Atriplex gmelinii C. A. Mey. Ex Bong._1838 is an annual halophytic herb found in East Asia and North America. The chloroplast genome of A. gmelinii was successfully sequenced. The assembled genome (151,852 bp; GC ratio, 37.3%) is composed of four subregions, with the large single copy (LSC; 83,872 bp; 35.4%) and small single copy (SSC; 17,812 bp; 30.9%) regions separated by two regions of inverted repeat regions (25,084 bp; 42.8%). A total of 130 genes were predicted with 85 protein-coding genes, 8 rRNAs, and 37 tRNAs. The phylogenetic analyses inferred from whole chloroplast genomes of 35 species, including 34 species in Amaranthaceae and one outgroup species, suggest a close relationship between A. gmelinii and A. centralasiatica.

Suppressive Effect of Bioactive Extracts of Bacillus sp. H8-1 and Bacillus sp. K203 on Tomato Wilt Caused by Clavibacter michiganensis subsp. michiganensis.

Tomatoes are cultivated worldwide, and are economically important. Clavibacter michiganensis subsp. michiganensis (Cmm) is a pathogen that causes canker and wilting in tomatoes, resulting in serious damage to tomato plants. We aimed to control Cmm proliferation using substances produced by useful microorganisms. The water extracts of strains H8-1 and K203 inhibited wilting caused by Cmm and slowed the pathogenic colonization in tomato plants. The relative expressions of celA, celB, pat1, and pelA of Cmm treated with the bacterial water extracts were reduced by 0.41-, 0.01-, 0.15-, and 0.14-fold for H8-1, respectively, and 0.45-, 0.02-, 0.13-, and 0.13-fold for K203, respectively, compared to controls at 72 h after treatments. In tomato plants inoculated with Cmm, when water extracts of H8-1 and K203 were treated, relative expression of ACO encoding 1-aminocyclopropane-1-carboxylic acid oxidase was suppressed by 0.26- and 0.23-fold, respectively, while PR1a was increased by 1.94- and 2.94-fold, respectively; PI2 expression was increased by 3.27-fold in water extract of H8-1-treated plants. As antioxidant enzymes of plants inoculated with Cmm, peroxidase and glutathione peroxidase levels were increased in K203-water-extract-treated plants, and catalase was increased in the case of the H8-1 water extract at 10 days after inoculation. In terms of soil enzyme activity, each water extract tended to increase urease activity and microbial diversity; in addition, K203 water extract increased plant growth. Thus, H8-1 and K203 water extracts can be used as potential biocontrol agents against Cmm.

Oxidative stress response and programmed cell death guided by NAC013 modulate pithiness in radish taproots.

Radish, Raphanus sativus L., is an important root crop that is cultivated worldwide. Owing to its evolutionary proximity to Arabidopsis thaliana, radish can be used as a model root crop in research on the molecular basis of agronomic traits. Pithiness is a significant defect that reduces the production of radish with commercial value; however, traditional breeding to eliminate this trait has thus far been unsuccessful. Here, we performed transcriptomics and genotype-by-sequencing (GBS)-based quantitative trait locus (QTL) analyses of radish inbred lines to understand the molecular basis of pithiness in radish roots. The transcriptome data indicated that pithiness likely stems from the response to oxidative stress, leading to cell death of the xylem parenchyma during the root-thickening process. Subsequently, we narrowed down a list of candidates responsible for pithiness near a major QTL and found polymorphisms in a radish homologue of Arabidopsis ANAC013 (RsNAC013), an endoplasmic reticulum bound NAC transcription factor that is targeted to the nucleus to mediate the mitochondrial retrograde signal. We analysed the effects of polymorphisms in RsNAC013 using Arabidopsis transgenic lines overexpressing RsNAC013 alleles as well as in radish inbred lines bearing these alleles. This analysis indicated that non-synonymous variations within the coding sequence result in different levels of RsNAC013 activities, thereby providing a genetic condition for root pithiness. The elevated oxidative stress or hypoxia that activates RsNAC013 for mitochondrial signalling enhances this process. Collectively, this study serves as an exemplary case of translational research taking advantage of the extensive information available from a model organism.

The Functional Association of ACQOS/VICTR with Salt Stress Resistance in Arabidopsis thaliana Was Confirmed by CRISPR-Mediated Mutagenesis.

Clustered regularly interspaced palindromic repeat (CRISPR)-mediated mutagenesis has become an important tool in plant research, enabling the characterization of genes via gene knock-out. CRISPR genome editing tools can be applied to generate multi-gene knockout lines. Typically, multiple single-stranded, single guide RNAs (gRNAs) must be expressed in an organism to target multiple genes simultaneously; however, a single gRNA can target multiple genes if the target genes share similar sequences. A gene cluster comprising ACQUIRED OSMOTOLERANCE (ACQOS; AT5G46520) and neighboring nucleotide-binding leucine-rich repeats (NLRs; AT5G46510) is associated with osmotic tolerance. To investigate the role of ACQOS and the tandemly arranged NLR in osmotic tolerance, we introduced small insertion/deletion mutations into two target genes using a single gRNA and obtained transformant plant lines with three different combinations of mutant alleles. We then tested our mutant lines for osmotic tolerance after a salt-stress acclimation period by determining the chlorophyll contents of the mutant seedlings. Our results strongly suggest that ACQOS is directly associated with salt resistance, while the neighboring NLR is not. Here, we confirmed previous findings suggesting the involvement of ACQOS in salt tolerance and demonstrated the usefulness of CRISPR-mediated mutagenesis in validating the functions of genes in a single genetic background.

The efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana.

CRISPR/Cas9-mediated genome editing is an important and versatile technology in modern biological research. Recent advancements include base-editing CRISPR tools that enable targeted nucleotide substitutions using a fusion protein comprising a nickase variant of Cas9 and a base deaminase. Improvements in base editing efficiencies and inheritable of edited loci need to be made to make CRISPR a viable system in plants. Here, we report efficiency of cytosine base editors (CBEs) in Arabidopsis thaliana by applying the strong endogenous RPS5a promoter to drive the expression of nickase Cas9 and either rAPOBEC1 from rat (BE3) or the PmCDA1 activation-induced cytidine deaminase from sea lamprey (AIDv2). Compared with the strong heterologous CaMV35S promoter of viral origin, the RPS5a promoter improved CBE efficiency by 32% points with the number of T1 plants showing over 50% conversion ratio when the LFY gene was targeted. CBE induced nonsense mutations in LFY via C-to-T conversion, which resulted in loss-of-function lfy phenotypes; defects in LFY function were associated with the targeted base substitutions. Our data suggest that optimal promoter choice for CBE expression may affect base-editing efficiencies in plants. The results provide a strategy to optimize low-efficiency base editors and demonstrate their applicability for functional assays and trait development in crop research.