Efficient tobacco rattle virus-induced gene editing in tomato mediated by the CRISPR/Cas9 system.

Plant virus-based sgRNA delivery strategy has been widely applied for efficient genome editing across various plant species, leveraging its significant advantages in the rapid expression and expansion of sgRNA through virus replication and movement. However, the efficacy of the virus-induced gene editing (VIGE) tool in tomato has yet to be explored. In this paper, we established a TRV-mediated CRISPR/Cas9 genome editing system in the somatic cells of tomato, reporting the validation of VIGE and evaluating the mutagenesis efficiency in both tomato leaves and fruits using high-throughput sequencing. The results demonstrated an approximate 65% efficiency of VIGE in tomato leaves for the selected target genes, with VIGE efficiency reaching up to 50% in tomato fruits. This research not only introduces an efficient tool for reverse genetics but also reveals substantial potential of VIGE in surpassing traditional tissue culture techniques for creating heritable mutations in tomato.

Analysis of the Positive Effects of Optimizing the Sequence of Attracting and Suctioning Below the Glottis on the Incidence of Ventilator-Associated Pneumonia.

Objective: This study aimed to investigate the impact of optimizing the subglottic suction and aspiration sequence on ventilator-associated pneumonia (VAP) incidence. Methods: A total of 108 patients undergoing transcatheter orotracheal intubation with subglottic secretion drainage (SSD) and mechanical ventilation were selected from the Department of Critical Care Medicine in our hospital between September 2021 and March 2023. The patients were randomly assigned to either the observation group or the control group (54 cases each) using a random number method. In the control group, patients underwent manual airway suction followed by subglottic suction with -100 mmHg pressure. In the observation group, subglottic suction with -100 mmHg pressure was performed first, followed by manual airway suction. The comparative analysis included blood gas parameters, sputum suction effectiveness, VAP occurrence, 28-day morbidity and mortality rates, tracheal secretion culture results, and the workload of nurses. Results: The observation group exhibited significantly shorter tape replacement time, sputum suction time, and number of suctions, along with a longer suction interval compared to the control group (P < 0.05). Post-suctioning, the observation group demonstrated improved blood gas function and a lower incidence of VAP (P < .05). No significant difference in adverse reaction incidence was observed between the two groups (P > .05); however, the Kolcaba score was higher in the observation group (P < .05). Conclusions: Pre-endotracheal intubation oral and nasal sputum suctioning proves effective in reducing the risk of VAP, lessening the workload of nurses, and enhancing the comfort of sputum suctioning.

Divergent Retention of Sucrose Metabolism Genes after Whole Genome Triplication in the Tomato (Solanum lycopersicum).

Sucrose, the primary carbon transport mode and vital carbohydrate for higher plants, significantly impacts plant growth, development, yield, and quality formation. Its metabolism involves three key steps: synthesis, transport, and degradation. Two genome triplication events have occurred in Solanaceae, which have resulted in massive gene loss. In this study, a total of 48 and 65 genes from seven sucrose metabolism gene families in Vitis vinifera and Solanum lycopersicum were identified, respectively. The number of members comprising the different gene families varied widely. And there were significant variations in the pattern of gene duplication and loss in the tomato following two WGD events. Tandem duplication is a major factor in the expansion of the SWEET and Acid INV gene families. All the genes are irregularly distributed on the chromosomes, with the majority of the genes showing collinearity with the grape, particularly the CIN family. And the seven gene families were subjected to a purifying selection. The expression patterns of the different gene families exhibited notable variations. This study presents basic information about the sucrose metabolism genes in the tomato and grape, and paves the way for further investigations into the impact of SCT events on the phylogeny, gene retention duplication, and function of sucrose metabolism gene families in the tomato or Solanaceae, and the adaptive evolution of the tomato.

Effects of Storage Temperature at the Early Postharvest Stage on the Firmness, Bioactive Substances, and Amino Acid Compositions of Chili Pepper (Capsicum annuum L.).

The commercial and nutritional quality of chili peppers deteriorates rapidly after harvest. So far, little is known about the effect of temperature on postharvest chili pepper quality. This study elucidated the effects of two temperatures (20 °C and 30 °C) on chili peppers' postharvest firmness, flavor, and nutritional attributes. We found that compared to 20 °C, 30 °C escalated the decline in fruit firmness, capsaicin content, and dihydrocapsaicin content, while enhancing the increment in water loss and electrical conductivity, as well as total carotenoids and ascorbic acid content. The contents of most amino acids (AAs) decreased significantly during postharvest storage compared to their initial values, whether stored at 20 °C or 30 °C; however, 30 °C had a more substantial impact than 20 °C. Meanwhile, as for soluble protein and amino acid compositions, the effect of storage temperature was genotype-dependent, as reflected by differential changes in total AA contents, single AA contents, essential AA ratio, delicious AA ratio, etc., under the 20 °C or 30 °C treatments. In conclusion, our findings reveal the influence of temperature on pepper quality, showing that the storage temperature of 20 °C was better for maintaining chili quality than 30 °C from the perspective of overall commercial attributes.

Analysis of the Influence of Waste Seashell as Modified Materials on Asphalt Pavement Performance.

An increasing amount of waste seashells in China has caused serious environmental pollution and resource waste. This paper aims to solve these problems by using waste seashells as modified materials to prepare high-performance modified asphalt. In this study, seashell powder (SP) and stratum corneum-exfoliated seashell powder (SCESP) were adopted to prepare 10%, 20% and 30% of seashell powder-modified asphalt (SPMA) and stratum corneum-exfoliated seashell powder-modified asphalt (SCESPMA) by the high-speed shear apparatus, respectively. The appearance and composition of two kinds of SPs were observed and determined by the scanning electron microscope (SEM). The types of functional groups, temperature frequency characteristics, low temperature performance and adhesion of SPMA were tested by the Fourier-transform infrared (FTIR) spectrometer, dynamic shear rheometer (DSR), bending beam rheometer (BBR) and contact angle meter. The results show that the SP and SCESP are rough and porous, and their main component is CaCO3, which is physically miscible to asphalt. When the loading frequency ranges from 0.1 Hz to 10 Hz, the complex shear modulus (G*) and phase angle (δ) of SPMA and SCESPMA increase and decrease, respectively. At the same load frequency, SCESPMA has a larger G* and a smaller δ than SPMA. At the same temperature, SCESPMA has a larger rutting factor (G*/sin δ) and better high-temperature deformation resistance than SPMA. SP and SCESP reduce the low-temperature cracking resistance of asphalt, of which SCESP has a more adverse effect on the low-temperature performance of asphalt than SP. When SP and SCESP are mixed with asphalt, the cohesion work (Waa), adhesion work (Was) and comprehensive evaluation parameters of water stability (ER1, ER2 and ER3) of asphalt are improved. It is shown that both SP and SCESP have good water damage resistance, of which SCESP has better water damage resistance than SP. These research results have important reference value for the application of waste biological materials in asphalt pavement.

Post-Harvest LED Light Irradiation Affects Firmness, Bioactive Substances, and Amino Acid Compositions in Chili Pepper (Capsicum annum L.).

Chili pepper is an important vegetable and spice crop with high post-harvest deteriorations in terms of commercial and nutritional quality. Light-emitting diodes (LEDs) are eco-friendly light sources with various light spectra that have been demonstrated to improve the shelf-life of various vegetables by manipulating light quality; however, little is known about their effects on the post-harvest nutritional quality of chili peppers. This study investigated the effects of different LED lightings on the post-harvest firmness and nutritional quality of chili peppers. We found that red and blue light could increase the content of capsaicinoids, whereas white and red light could increase the essential and aromatic amino acid (AA) content in pepper. Nonetheless, the influence of light treatments on AA contents and compositions depends strongly on the pepper genotype, which was reflected by total AA content, single AA content, essential AA ratio, delicious AA ratio, etc., that change under different light treatments. Additionally, light affected fruit firmness and the content of nutrients such as chlorophyll, vitamin C, and total carotenoids, to varying degrees, depending on pepper genotypes. Thus, our findings indicate that LED-light irradiation is an efficient and promising strategy for preserving or improving the post-harvest commercial and nutritional quality of pepper fruit.

Comprehensive Analysis of Subcellular Localization, Immune Function and Role in Bacterial wilt Disease Resistance of Solanum lycopersicum Linn. ROP Family Small GTPases.

ROPs (Rho-like GTPases from plants) belong to the Rho-GTPase subfamily and serve as molecular switches for regulating diverse cellular events, including morphogenesis and stress responses. However, the immune functions of ROPs in Solanum lycopersicum Linn. (tomato) is still largely unclear. The tomato genome contains nine genes encoding ROP-type small GTPase family proteins (namely SlRop1-9) that fall into five distinct groups as revealed by phylogenetic tree. We studied the subcellular localization and immune response induction of nine SlRops by using a transient overexpression system in Nicotiana benthamiana Domin. Except for SlRop1 and SlRop3, which are solely localized at the plasma membrane, most of the remaining ROPs have additional nuclear and/or cytoplasmic distributions. We also revealed that the number of basic residues in the polybasic region of ROPs tends to be correlated with their membrane accumulation. Though nine SlRops are highly conserved at the RHO (Ras Homology) domains, only seven constitutively active forms of SlRops were able to trigger hypersensitive responses. Furthermore, we analyzed the tissue-specific expression patterns of nine ROPs and found that the expression levels of SlRop3, 4 and 6 were generally high in different tissues. The expression levels of SlRop1, 2 and 7 significantly decreased in tomato seedlings after infection with Ralstonia solanacearum (E.F. Smith) Yabuuchi et al. (GMI1000); the others did not respond. Infection assays among nine ROPs showed that SlRop3 and SlRop4 might be positive regulators of tomato bacterial wilt disease resistance, whereas the rest of the ROPs may not contribute to defense. Our study provides systematic evidence of tomato Rho-related small GTPases for localization, immune response, and disease resistance.

Molecular Evolution and Functional Divergence of Stress-Responsive Cu/Zn Superoxide Dismutases in Plants.

Superoxide dismutases (SODs), a family of antioxidant enzymes, are the first line of defense against oxidative damage and are ubiquitous in every cell of all plant types. The Cu/Zn SOD, one of three types of SODs present in plant species, is involved in many of the biological functions of plants in response to abiotic and biotic stresses. Here, we carried out a comprehensive analysis of the Cu/Zn SOD gene family in different plant species, ranging from lower plants to higher plants, and further investigated their organization, sequence features, and expression patterns in response to biotic and abiotic stresses. Our results show that plant Cu/Zn SODs can be divided into two subfamilies (group I and group II). Group II appeared to be conserved only as single- or low-copy genes in all lineages, whereas group I genes underwent at least two duplication events, resulting in multiple gene copies and forming three different subgroups (group Ia, group Ib, and group Ic). We also found that, among these genes, two important events-the loss of introns and the loss of and variation in signal peptides-occurred over the long course of their evolution, indicating that they were involved in shifts in subcellular localization from the chloroplast to cytosol or peroxisome and underwent functional divergence. In addition, expression patterns of Cu/Zn SOD genes from Arabidopsis thaliana and Solanum lycopersicum were tested in different tissues/organs and developmental stages and under different abiotic stresses. The results indicate that the Cu/Zn SOD gene family possesses potential functional divergence and may play vital roles in ROS scavenging in response to various stresses in plants. This study will help establish a foundation for further understanding these genes' function during stress responses.

Three highly conserved hydrophobic residues in the predicted α2-helix of rice NLR protein Pit contribute to its localization and immune induction.

Nucleotide-binding leucine-rich repeat (NLR) proteins work as crucial intracellular immune receptors. N-terminal domains of NLRs fall into two groups, coiled-coil (CC) and Toll-interleukin 1 receptor domains, which play critical roles in signal transduction and disease resistance. However, the activation mechanisms of NLRs, and how their N-termini function in immune induction, remain largely unknown. Here, we revealed that the CC domain of a rice NLR Pit contributes to self-association. The Pit CC domain possesses three conserved hydrophobic residues that are known to be involved in oligomer formation in two NLRs, barley MLA10 and Arabidopsis RPM1. Interestingly, the function of these residues in Pit differs from that in MLA10 and RPM1. Although three hydrophobic residues are important for Pit-induced disease resistance against rice blast fungus, they do not participate in self-association or binding to downstream signalling molecules. By homology modelling of Pit using the Arabidopsis ZAR1 structure, we tried to clarify the role of three conserved hydrophobic residues and found that they are located in the predicted α2-helix of the Pit CC domain and involved in the plasma membrane localization. Our findings provide novel insights for understanding the mechanisms of NLR activation as well as the relationship between subcellular localization and immune induction.

Damage Induced to DNA and Its Constituents by 0-3 eV UV Photoelectrons.

The complex physical and chemical interactions between DNA and 0-3 eV electrons released by UV photoionization can lead to the formation of various lesions such as base modifications and cleavage, crosslinks and single strand breaks. Furthermore, in the presence of platinum chemotherapeutic agents, these electrons can cause clustered lesions, including double strand breaks. We explain the mechanisms responsible for these damages via the production 0-3 eV electrons by UVC radiation, and by UV photons of any wavelengths, when they are produced by photoemission from nanoparticles lying within about 10 nm from DNA. We review experimental evidence showing that a single 0-3 eV electron can produce these damages. The foreseen benefits UV-irradiation of nanoparticles targeted to the cell nucleus are mentioned in the context of cancer therapy, as well as the potential hazards to human health when they are present in cells.

Light regulation of potassium in plants.

Essential macronutrient potassium (K) and environmental signal light regulate a number of vital plant biological processes related to growth, development, and stress response. Recent research has shown connections between the perception of light and the regulation of K in plants. Photoreceptors-mediated wavelength-specific light perception activates signaling cascades which mediate stomatal movement by altering K+influx/efflux via K+ channels in the guard cells. The quality, intensity, and duration of light affect the regulation of K nutrition and crop quality. Blue/red illumination or red combined blue light treatment increases the expression levels of K transporter genes, K uptake and accumulation, leading to increased lycopene synthesis and improved fruit color in tomato. Despite the commonalities of light and K in multiple functions, our understanding of light regulation of K and associated physiological and molecular processes is fragmentary. In this review, we take a look at the light-controlled K uptake and utilization in plants and propose working models to show potential mechanisms. We discuss major light signaling components, their possible involvement in K nutrition, stomatal movement and crop quality by linking the perception of light signal and subsequent regulation of K. We also pose some outstanding questions to guide future research. Our analysis suggests that the enhancement of K utilization efficiency by manipulation of light quality and light signaling components can be a promising strategy for K management in crop production.

ELONGATED HYPOCOTYL 5 mediates blue light-induced starch degradation in tomato.

Starch is the major storage carbohydrate in plants, and its metabolism in chloroplasts depends mainly on light. However, the mechanism through which photoreceptors regulate starch metabolism in chloroplasts is unclear. In this study, we found that the cryptochrome 1a (CRY1a)-mediated blue light signal is critical for regulating starch accumulation by inducing starch degradation through the transcription factor HY5 in chloroplasts in tomato. cry1a mutants and HY5-RNAi plants accumulated more starch and presented lower transcript levels of starch degradation-related genes in their leaves than wild-type plants. Blue light significantly induced the transcription of starch degradation-related genes in wild-type and CRY1a- or HY5-overexpressing plants but had little effect in cry1a and HY5-RNAi plants. Dual-luciferase assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation-qPCR revealed that HY5 could activate the starch degradation-related genes PWD, BAM1, BAM3, BAM8, MEX1, and DPE1 by directly binding to their promoters. Silencing of HY5 and these starch degradation-related genes in CRY1a-overexpressing plants led to increased accumulation of starch and decreased accumulation of soluble sugars. The findings presented here not only deepen our understanding of how light controls starch degradation and sugar accumulation but also allow us to explore potential targets for improving crop quality.

The tomato 2-oxoglutarate-dependent dioxygenase gene SlF3HL is critical for chilling stress tolerance.

Low temperature is a major stress that severely affects plant development, growth, distribution, and productivity. Here, we examined the function of a 2-oxoglutarate-dependent dioxygenase-encoding gene, SlF3HL, in chilling stress responses in tomato (Solanum lycopersicum cv. Alisa Craig [AC]). Knockdown (KD) of SlF3HL (through RNA interference) in tomato led to increased sensitivity to chilling stress as indicated by elevated levels of electrolyte leakage, malondialdehyde (MDA) and reactive oxygen species (ROS). In addition, the KD plants had decreased levels of proline and decreased activities of peroxisome and superoxide dismutase. The expression of four cold-responsive genes was substantially reduced in the KD plants. Furthermore, seedling growth was significantly greater in AC or SlF3HL-overexpression plants than in the KD plants under either normal growth conditions with methyl jasmonate (MeJA) or chilling stress conditions. SlF3HL appears to positively regulate JA accumulation and the expression of JA biosynthetic and signaling genes under chilling stress. Together, these results suggest that SlF3HL is a positive regulator of chilling stress tolerance and functions in the chilling stress tolerance pathways, possibly by regulating JA biosynthesis, JA signaling, and ROS levels.

TiO2 Nanotubes/Ag/MoS2 Meshy Photoelectrode with Excellent Photoelectrocatalytic Degradation Activity for Tetracycline Hydrochloride.

A novel type of TiO2 nanotubes (NTs)/Ag/MoS2 meshy photoelectrode was fabricated with highly oriented TiO2 nanotube arrays grown from a Ti mesh supporting Ag nanoparticles and three-dimensional MoS2 nanosheets. In this structure, Ag nanoparticles act as bridges to connect MoS2 and TiO2 and pathways for electron transfer, ensuring the abundant production of active electrons, which are the source of •O2-. The TiO2 NTs/Ag/MoS2 mesh can be used as both photocatalyst and electrode, exhibiting enhanced photoelectrocatalytic efficiency in degrading tetracycline hydrochloride under visible light irradiation (λ ≥ 420 nm). Compared to unmodified TiO2 NTs, the improved photoelectrocatalytic activity of the TiO2 NTs/Ag/MoS2 arise from the formation of Z-scheme heterojunctions, which facilitate the efficient separation of photogenerated electron-hole pairs through the Schottky barriers at the interfaces of TiO2 NTs⁻Ag and Ag⁻MoS2.

The bZip transcription factor HY5 mediates CRY1a-induced anthocyanin biosynthesis in tomato.

The production of anthocyanin is regulated by light and corresponding photoreceptors. In this study, we found that exposure to blue light and overexpression of CRY1a are associated with increased accumulation of anthocyanin in tomato (Solanum lycopersicum L.). These responses are the result of changes in mRNA and the protein levels of SlHY5, which is a transcription factor. In vitro and in vivo experiments using electrophoretic mobility shift assay and ChIP-qPCR assays revealed that SlHY5 could directly recognize and bind to the G-box and ACGT-containing element in the promoters of anthocyanin biosynthesis genes, such as chalcone synthase 1, chalcone synthase 2, and dihydroflavonol 4-reductase. Silencing of SlHY5 in OE-CRY1a lines decreased the accumulation of anthocyanin. The findings presented here not only deepened our understanding of how light controls anthocyanin biosynthesis and associated photoprotection in tomato leaves, but also allowed us to explore potential targets for improving pigment production.

Tomato CRY1a plays a critical role in the regulation of phytohormone homeostasis, plant development, and carotenoid metabolism in fruits.

Blue light photoreceptors, cryptochromes (CRYs), regulate multiple aspects of plant growth and development. However, our knowledge of CRYs is predominantly based on model plant Arabidopsis at early growth stage. In this study, we elucidated functions of CRY1a gene in mature tomato (Solanum lycopersicum) plants by using cry1a mutants and CRY1a-overexpressing lines (OE-CRY1a-1 and OE-CRY1a-2). In comparison with wild-type plants, cry1a mutants are relatively tall, accumulate low biomass, and bear more fruits, whereas OE-CRY1a plants are short stature, and they not only flower lately but also bear less fruits. RNA-seq, qRT-PCR, and LC-MS/MS analysis revealed that biosynthesis of gibberellin, cytokinin, and jasmonic acid was down-regulated by CRY1a. Furthermore, DNA replication was drastically inhibited in leaves of OE-CRY1a lines, but promoted in cry1a mutants with concomitant changes in the expression of cell cycle genes. However, CRY1a positively regulated levels of soluble sugars, phytofluene, phytoene, lycopene, and ß-carotene in the fruits. The results indicate the important role of CRY1a in plant growth and have implications for molecular interventions of CRY1a aimed at improving agronomic traits.

DWARF overexpression induces alteration in phytohormone homeostasis, development, architecture and carotenoid accumulation in tomato.

Brassinosteroids (BRs) play a critical role in plant growth, development and stress response; however, genetic evidence for the BR-mediated integrated regulation of plant growth still remains elusive in crop species. Here, we clarified the function of DWARF (DWF), the key BR biosynthetic gene in tomato, in the regulation of plant growth and architecture, phytohormone homeostasis and fruit development by comparing wild type, d^(im), a weak allele mutant impaired in DWF, and DWF-overexpressing plants in tomato. Results showed that increases in DWF transcripts and endogenous BR level resulted in improved germination, lateral root development, CO2 assimilation and eventually plant growth as characterized by slender and compact plant architecture. However, an increase in DWF transcript down-regulated the accumulation of gibberellin, which was associated with decreases in leaf size and thickness. BRs positively regulated lateral bud outgrowth, which was associated with decreased transcript of Aux/IAA3, and the ethylene-dependent petiole bending and fruit ripening. Notably, overexpression of DWF did not significantly alter fruit yield per plant; however, increases by 57.4% and 95.3% might be estimated in fruit yield per square metre in two transgenic lines due to their compact architecture. Significantly, BR level was positively related with the carotenoid accumulation in the fruits. Taken together, our results demonstrate that BRs are actively involved in the regulation of multiple developmental processes relating to agronomical important traits.

A clinical microchip for evaluation of single immune cells reveals high functional heterogeneity in phenotypically similar T cells.

Cellular immunity has an inherent high level of functional heterogeneity. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. We report a microfluidic platform designed for highly multiplexed (more than ten proteins), reliable, sample-efficient (∼1 × 10(4) cells) and quantitative measurements of secreted proteins from single cells. We validated the platform by assessment of multiple inflammatory cytokines from lipopolysaccharide (LPS)-stimulated human macrophages and comparison to standard immunotechnologies. We applied the platform toward the ex vivo quantification of T cell polyfunctional diversity via the simultaneous measurement of a dozen effector molecules secreted from tumor antigen-specific cytotoxic T lymphocytes (CTLs) that were actively responding to tumor and compared against a cohort of healthy donor controls. We observed profound, yet focused, functional heterogeneity in active tumor antigen-specific CTLs, with the major functional phenotypes quantitatively identified. The platform represents a new and informative tool for immune monitoring and clinical assessment.

Integrated barcode chips for rapid, multiplexed analysis of proteins in microliter quantities of blood.

As the tissue that contains the largest representation of the human proteome, blood is the most important fluid for clinical diagnostics. However, although changes of plasma protein profiles reflect physiological or pathological conditions associated with many human diseases, only a handful of plasma proteins are routinely used in clinical tests. Reasons for this include the intrinsic complexity of the plasma proteome, the heterogeneity of human diseases and the rapid degradation of proteins in sampled blood. We report an integrated microfluidic system, the integrated blood barcode chip that can sensitively sample a large panel of protein biomarkers over broad concentration ranges and within 10 min of sample collection. It enables on-chip blood separation and rapid measurement of a panel of plasma proteins from quantities of whole blood as small as those obtained by a finger prick. Our device holds potential for inexpensive, noninvasive and informative clinical diagnoses, particularly in point-of-care settings.