Super-pangenome analyses highlight genomic diversity and structural variation across wild and cultivated tomato species.

Effective utilization of wild relatives is key to overcoming challenges in genetic improvement of cultivated tomato, which has a narrow genetic basis; however, current efforts to decipher high-quality genomes for tomato wild species are insufficient. Here, we report chromosome-scale tomato genomes from nine wild species and two cultivated accessions, representative of Solanum section Lycopersicon, the tomato clade. Together with two previously released genomes, we elucidate the phylogeny of Lycopersicon and construct a section-wide gene repertoire. We reveal the landscape of structural variants and provide entry to the genomic diversity among tomato wild relatives, enabling the discovery of a wild tomato gene with the potential to increase yields of modern cultivated tomatoes. Construction of a graph-based genome enables structural-variant-based genome-wide association studies, identifying numerous signals associated with tomato flavor-related traits and fruit metabolites. The tomato super-pangenome resources will expedite biological studies and breeding of this globally important crop.

UPLC-MS/MS Assay for Quantification of Wedelolactone and Demethylwedelolactone in Rat Plasma and the Application to a Preclinical Pharmacokinetic Study.

AIMS AND OBJECTIVE: Wedelolactone and demethylwedelolactone are the two major coumarin constituents of Herba Ecliptae. The objective of this work was to develop and validate a sensitive, rapid, and robust UPLC-MS/MS method for the simultaneous quantification of wedelolactone and demethylwedelolactone in rat plasma. MATERIALS AND METHODS: Wedelolactone and demethylwedelolactone were extracted from rat plasma by protein precipitation with acetonitrile. Electrospray ionization in negative mode and selected reaction monitoring (SRM) were used for wedelolactone and demethylwedelolactone at the transitions m/z 312.8→298.0 and m/z 299.1→270.6, respectively. Chromatographic separation was conducted on a Venusil C18 column (50 mm × 2.1 mm, 5 µm) with isocratic elution of acetonitrile-0.1% formic acid in water (55:45, v/v) at a flow rate of 0.3 mL/min. A linear range was observed over the concentration range of 0.25-100 ng/mL for wedelolactone and demethylwedelolactone. RESULTS: They reached their maximum plasma concentrations (Cmax, 74.9±13.4 ng/mL for wedelolactone and 41.3±9.57 ng/mL for demethylwedelolactone) at the peak time (Tmax) of 0.633 h and 0.800 h, respectively. The AUC0-t value of wedelolactone (260.8±141.8 ng h/mL) was higher than that of demethylwedelolactone (127.4±52.7 ng h/mL) by approximately 2-fold, whereas the terminal elimination half-life (t1/2) of wedelolactone (2.20±0.59 h) showed the approximately same as that of demethylwedelolactone (2.08±0.69 h). CONCLUSION: Based on full validation according to US FDA guidelines, this UPLC-MS/MS method was successfully applied to a pharmacokinetic study in rats.

iTRAQ-Based Proteomics Reveals that the Tomato ms1035 Gene Causes Male Sterility through Compromising Fat Acid Metabolism.

So far, over 50 spontaneous male sterile mutants of tomato have been described and most of them are categorized as genetic male sterility. To date, the mechanism of tomato genetic male sterility remained unclear. In this study, differential proteomic analysis is performed between genetic male sterile line (2-517), which carries the male sterility (ms1035 ) gene, and its wild-type (VF-11) using isobaric tags for relative and absolute quantification-based strategy. A total of 8272 proteins are quantified in the 2-517 and VF-11 lines at the floral bud and florescence stages. These proteins are involved in different cellular and metabolic processes, which express obvious functional tendencies toward the hydroxylation of the ω-carbon in fatty acids, the tricarboxylic acid cycle, the glycolytic, and pentose phosphate pathways. Based on the results, a protein network explaining the mechanisms of tomato genetic male sterility is proposed, finding the compromising fat acid metabolism may cause the male sterility. These results are confirmed by parallel reaction monitoring, quantitative Real-time PCR (qRT-PCR), and physiological assays. Taken together, these results provide new insights into the metabolic pathway of anther abortion induced by ms1035 and offer useful clues to identify the crucial proteins involved in genetic male sterility in tomato.

Knockdown of CaHSP60-6 confers enhanced sensitivity to heat stress in pepper (Capsicum annuum L.).

MAIN CONCLUSION: HSP60 gene family in pepper was analyzed through bioinformatics along with transcriptional regulation against multiple abiotic and hormonal stresses. Furthermore, the knockdown of CaHSP60-6 increased sensitivity to heat stress. The 60 kDa heat shock protein (HSP60) also known as chaperonin (cpn60) is encoded by multi-gene family that plays an important role in plant growth, development and in stress response as a molecular chaperone. However, little is known about the HSP60 gene family in pepper (Capsicum annuum L.). In this study, 16 putative pepper HSP60 genes were identified through bioinformatic tools. The phylogenetic tree revealed that eight of the pepper HSP60 genes (50%) clustered into group I, three (19%) into group II, and five (31%) into group III. Twelve (75%) CaHSP60 genes have more than 10 introns, while only a single gene contained no introns. Chromosomal mapping revealed that the tandem and segmental duplication events occurred in the process of evolution. Gene ontology enrichment analysis predicted that CaHSP60 genes were responsible for protein folding and refolding in an ATP-dependent manner in response to various stresses in the biological processes category. Multiple stress-related cis-regulatory elements were found in the promoter region of these CaHSP60 genes, which indicated that these genes were regulated in response to multiple stresses. Tissue-specific expression was studied under normal conditions and induced under 2 h of heat stress measured by RNA-Seq data and qRT-PCR in different tissues (roots, stems, leaves, and flowers). The data implied that HSP60 genes play a crucial role in pepper growth, development, and stress responses. Fifteen (93%) CaHSP60 genes were induced in both, thermo-sensitive B6 and thermo-tolerant R9 lines under heat treatment. The relative expression of nine representative CaHSP60 genes in response to other abiotic stresses (cold, NaCl, and mannitol) and hormonal applications [ABA, methyl jasmonate (MeJA), and salicylic acid (SA)] was also evaluated. Knockdown of CaHSP60-6 increased the sensitivity to heat shock treatment as documented by a higher relative electrolyte leakage, lipid peroxidation, and reactive oxygen species accumulation in silenced pepper plants along with a substantial lower chlorophyll content and antioxidant enzyme activity. These results suggested that HSP60 might act as a positive regulator in pepper defense against heat and other abiotic stresses. Our results provide a basis for further functional analysis of HSP60 genes in pepper.

Validation and application of a novel LC/MS/MS method for the determination of isoginkgetin in rat plasma.

Isoginkgetin is a biflavonoid compound isolated from the leaf extracts of Ginkgo biloba. In this study, an liquid chromatography-tandem mass spectrometry (LC/MS/MS) with liquid-liquid extraction was developed and validated for the analysis of isoginkgetin in rat plasma. In the process of chromatographic separation, selected reaction monitoring transitions for isoginkgetin and IS were m/z 566.8 → 134.7 and m/z 430.8 → 269.3, respectively. The validation parameters including selectivity, linearity, LLOQ, accuracy, precision, matrix effect, stability and recovery were satisfactory. The intra- and inter-batch precision (RSD) were <12.1% in plasma, while the accuracy (RE) was within ±14.3%. This method was employed in a pharmacokinetic study on rats after the intravenous administration of isoginkgetin.

A small heat shock protein CaHsp25.9 positively regulates heat, salt, and drought stress tolerance in pepper (Capsicum annuum L.).

Extreme environmental conditions seriously affect crop growth and development, resulting in a decrease in crop yield and quality. However, small heat shock proteins (Hsp20s) play an important role in helping plants to avoid these negative impacts. In this study, we identified the expression pattern of the CaHsp25.9 gene in a thermo-tolerance pepper line R9 and thermo-sensitive line B6. The transcription of CaHsp25.9 was strongly induced by heat stress in both R9 and B6. The expression of CaHsp25.9 was induced by salt and drought stress in R9. Additionally, the CaHsp25.9 protein was localized in the cell membrane and cytoplasm. When silencing the CaHsp25.9 gene in the R9 line, the accumulation of malonaldehyde (MDA), relative electrolytic leakage, hydrogen peroxide, superoxide anion were increased, while total chlorophyll decreased under heat, salt, and drought stress. Over-expression of CaHsp25.9 in Arabidopsis resulted in decreased MDA, while proline, superoxide dismutase activity, germination, and root length increased under heat, salt, and drought stress. However, peroxidase activity was higher in drought stress but lower in heat and salt stress in transgenic Arabidopsis compared to the wild type (WT). Furthermore, the transcription of stress related genes was more highly induced in transgenic lines than WT. Our results indicated that CaHsp25.9 confers heat, salt, and drought stress tolerance to plants by reducing the accumulation of reactive oxygen species, enhancing the activity of antioxidant enzymes, and regulating the expression of stress-related genes. Therefore, these results may provide insight into plant adaption mechanisms developed in variable environments.

CaHSP16.4, a small heat shock protein gene in pepper, is involved in heat and drought tolerance.

Environmental stress affects growth and development of crops, and reduces yield and quality of crops. To cope with environmental stressors, plants have sophisticated defense mechanisms, including the HSF/HSP pathway. Here, we identify the expression pattern of CaHSP16.4 in thermo-tolerant and thermo-sensitive pepper (Capsicum annuum L.) lines. Under heat stress, R9 thermo-tolerant line had higher CaHSP16.4 expression level than the B6 thermo-sensitive line. Under drought stress, expression pattern of CaHSP16.4 was dynamic. Initially, CaHSP16.4 was downregulated then CaHSP16.4 significantly increased. Subcellular localization assay showed that CaHSP16.4 localizes in cytoplasm and nucleus. In the R9 line, silencing of CaHSP16.4 resulted in a significant increase in malonaldehyde content and a significant reduction in total chlorophyll content, suggesting that silencing of CaHSP16.4 reduces heat and drought stresses tolerance. Overexpression of CaHSP16.4 enhances tolerance to heat stress in Arabidopsis. Under heat stress, the survival rate of CaHSP16.4 overexpression lines was significantly higher than wild type. Furthermore, under heat, drought, and combined stress conditions, the CaHSP16.4-overexpression lines had lower relative electrolytic leakage and malonaldehyde content, higher total chlorophyll content, and higher activity levels of superoxide dismutase, catalase, ascorbic acid peroxidase, and glutathione peroxidase compared to wild type. Furthermore, the expression levels of the stress response genes in the overexpression lines were higher than the wild type. These results indicate that the overexpression of CaHSP16.4 enhances the ability of reactive oxygen species scavenging under heat and drought stress.

Determination of tubuloside B by LC-MS/MS and its application to a pharmacokinetic study in rats.

Tubuloside B, a novel neuroprotective phenylethanoid, is a major active constituent of Cistanche tubulosa and Cistanche deserticola. A specific and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantification of tubuloside B in rat plasma. Sample preparation was conducted through a protein-precipitation extraction with methanol using tubuloside A as internal standard (IS). Chromatographic separation was achieved using a Capcell Pak C18 column (2.0 × 50 mm, 5 µm) with a mobile phase of methanol-10 mm ammonium acetate buffer (70:30, v/v) in an isocratic elution. Mass spectrometry analysis was performed in negative ionization mode with selected reaction monitoring transitions at m/z 665.1 → 160.9 for tubuloside B, and m/z 827.1 → 160.9 for IS. Calibration curves were linear over the range of 1.64-1640 ng/mL for plasma samples samples (R2 > 0.990). The lower limit of quantification (LLOQ) was 1.64 ng/mL. The intra- and inter-day accuracy was between 92.3 and 113.0% with the RSD <9.23% at all LLOQ and quality control levels. Finally, this method was successfully applied in the pharmacokinetics study of tubuloside B after intravenous administration.

CRISPR/Cas9-induced Targeted Mutagenesis and Gene Replacement to Generate Long-shelf Life Tomato Lines.

Quickly and precisely gain genetically enhanced breeding elites with value-adding performance traits is desired by the crop breeders all the time. The present of gene editing technologies, especially the CRISPR/Cas9 system with the capacities of efficiency, versatility and multiplexing provides a reasonable expectation towards breeding goals. For exploiting possible application to accelerate the speed of process at breeding by CRISPR/Cas9 technology, in this study, the Agrobacterium tumefaciens-mediated CRISPR/Cas9 system transformation method was used for obtaining tomato ALC gene mutagenesis and replacement, in absence and presence of the homologous repair template. The average mutation frequency (72.73%) and low replacement efficiency (7.69%) were achieved in T0 transgenic plants respectively. None of homozygous mutation was detected in T0 transgenic plants, but one plant carry the heterozygous genes (Cas9/*-ALC/alc) was stably transmitted to T1 generations for segregation and genotyping. Finally, the desired alc homozygous mutants without T-DNA insertion (*/*-alc/alc) in T1 generations were acquired and further confirmed by genotype and phenotype characterization, with highlight of excellent storage performance, thus the recessive homozygous breeding elites with the character of long-shelf life were generated. Our results support that CRISPR/Cas9-induced gene replacement via HDR provides a valuable method for breeding elite innovation in tomato.