Comparison of Nutritional Diversity in Five Fresh Legumes Using Flavonoids Metabolomics and Postharvest Botrytis cinerea Defense Analysis of Peas Mediated by Sakuranetin.

Legumes possess several bioactive nutrients, including flavonoids, and the study of the flavonoid profile of legumes is of great significance to human health. Using widely targeted metabolomics, we revealed the flavonoid profiles of five popular fresh legumes: cowpea, soybean, pea, fava bean, and kidney bean. A total of 259 flavonoids were identified, and the flavonoid accumulation patterns of the five legumes were remarkably different. In addition to analyzing common and species-specific flavonoids in the five legumes, we also generalized representative flavonoids of various subclasses. We related these to the health-promoting effects of legumes. Furthermore, legumes' total flavonoid content and antioxidant system activity were also detected. Intriguingly, sakuranetin, the sole flavonoid phytoalexin that can be induced by UV radiation, was detected only in the peas by metabolomics. Meanwhile, we found that UV treatment could significantly increase the sakuranetin content and the postharvest Botrytis cinerea resistance of pea pods. This study provides clues for the target diet, industrial development of legumes, and a new idea for the postharvest preservation of peas.

Biofortified Rice Provides Rich Sakuranetin in Endosperm.

Sakuranetin plays a key role as a phytoalexin in plant resistance to biotic and abiotic stresses, and possesses diverse health-promoting benefits. However, mature rice seeds do not contain detectable levels of sakuranetin. In the present study, a transgenic rice plant was developed in which the promoter of an endosperm-specific glutelin gene OsGluD-1 drives the expression of a specific enzyme naringenin 7-O-methyltransferase (NOMT) for sakuranetin biosynthesis. The presence of naringenin, which serves as the biosynthetic precursor of sakuranetin made this modification feasible in theory. Liquid chromatography tandem mass spectrometry (LC-MS/MS) validated that the seeds of transgenic rice accumulated remarkable sakuranetin at the mature stage, and higher at the filling stage. In addition, the panicle blast resistance of transgenic rice was significantly higher than that of the wild type. Specially, the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging was performed to detect the content and spatial distribution of sakuranetin and other nutritional metabolites in transgenic rice seeds. Notably, this genetic modification also did not change the nutritional and quality indicators such as soluble sugars, total amino acids, total flavonoids, amylose, total protein, and free amino acid content in rice. Meanwhile, the phenotypes of the transgenic plant during the whole growth and developmental periods and agricultural traits such as grain width, grain length, and 1000-grain weight exhibited no significant differences from the wild type. Collectively, the study provides a conceptual advance on cultivating sakuranetin-rich biofortified rice by metabolic engineering. This new breeding idea may not only enhance the disease resistance of cereal crop seeds but also improve the nutritional value of grains for human health benefits.

Searching for the Virulence-contributing Genes of the Magnaporthe oryzae by Transcriptome Analysis.

Magnaporthe oryzae is a fungal pathogen that causes rice blast. Plant metabolites such as plant hormones and phytoalexin can promote or inhibit the rice blast infection. To study the effect of plant metabolites on M. oryzae, we selected salicylic acid (SA), abscisic acid (ABA), and a phytoalexin sakuranetin to treat M. oryzae grown on the medium. Through the analysis of transcriptome data, 185 and 38 genes, 803 and 156 genes, and 1525 and 428 genes were up- or down-regulated after SA, ABA, or sakuranetin treatment. Among these differentially expressed genes (DEGs), most of them were annotated to the cellular process and metabolic process in the biological process category and binding and catalytic activity in the molecular function category by GO analysis. According to KEGG pathway analysis, metabolism is the pathway with the highest number of DEGs, and the main enriched pathway is carbohydrate, lipid, and amino acid metabolism. In addition, we also found two ABA-induced up-regulated genes that may contribute to M. oryzae infection from the transcriptome data. We verified their expressions in M. oryzae that infected rice.

Pharmacokinetic Modeling to Guide Preclinical Development of an Islatravir-Eluting Reservoir-Style Biodegradable Implant for Long-Acting HIV PrEP.

Long-acting injectable cabotegravir is more effective than daily oral PrEP at preventing HIV transmission due to improved adherence, but requires bi-monthly large-volume intramuscular injections. Subcutaneous (SC) contraceptive implants can be formulated with antiretrovirals for extended-duration HIV PrEP. Islatravir (ISL) is a first-in-class, investigational antiretroviral with pharmacologic properties well-suited for implant delivery. We performed preclinical studies for the development of a reservoir-style, poly(ε-caprolactone) ISL-eluting implant by conducting a single-dose SC ISL dose-ranging pharmacokinetic (PK) study of 0.1, 0.3, and 1 mg/kg in adult Wistar rats. Non-compartmental analysis was conducted, and dose proportionality assessed for ISL plasma and intracellular islatravir-triphosphate (ISL-tp). Population PK models estimated ISL's unit impulse response to deconvolve ISL-implant in vivo absorption rate (mg/day) and cumulative mass (mg) from published rat plasma PK (n = 10). Drug release was interpreted using four kinetic models. Dose proportionality was affirmed for ISL and ISL-tp. A first-order, two-compartment model fitted the SC ISL bolus data. Mean (SD) absorption rate from 0 to 154 days was 0.072 ± 0.024 mg/day, and cumulative mass at 154 days was 8.67 ± 3.22 mg. ISL absorption was well-described by zero-order (r2 = 0.95) and Ritger-Peppas (r2 = 0.98). Our zero-order ISL-release poly(ε-caprolactone) implant is projected to achieve clinical PK above ISL-tp's PrEP efficacy threshold. Continued development for HIV PrEP applications is warranted.

The OsBZR1-OsSPX1/2 module fine-tunes the growth-immunity trade-off in adaptation to phosphate availability in rice.

The growth-promoting hormones brassinosteroids (BRs) and their key signaling component BZR1 play a vital role in balancing normal growth and defense reactions. Here, we discovered that BRs and OsBZR1 upregulated sakuranetin accumulation and conferred basal defense against Magnaporthe oryzae infection under normal conditions. Resource shortages, including phosphate (Pi) deficiency, potentially disrupt this growth-defense balance. OsSPX1 and OsSPX2 have been reported to sense Pi concentration and interact with the Pi signal mediator OsPHR2, thus regulating Pi starvation responses. In this study, we discovered that OsSPX1/2 interacts with OsBZR1 in both Pi-sufficient and Pi-deficient conditions, inhibiting BR-responsive genes. When Pi is sufficient, OsSPX1/2 is captured by OsPHR2, enabling most of OsBZR1 to promote plant growth and maintain basal resistance. In response to Pi starvation, more OsSPX1/2 is released from OsPHR2 to inhibit OsBZR1 activity, resulting in slower growth. Collectively, our study reveals that the OsBZR1-SPX1/2 module balances the plant growth-immunity trade-off in response to Pi availability.

Pharmacokinetic Study of Islatravir and Etonogestrel Implants in Macaques.

The prevention of HIV and unintended pregnancies is a public health priority. Multi-purpose prevention technologies capable of long-acting HIV and pregnancy prevention are desirable for women. Here, we utilized a preclinical macaque model to evaluate the pharmacokinetics of biodegradable ε-polycaprolactone implants delivering the antiretroviral islatravir (ISL) and the contraceptive etonogestrel (ENG). Three implants were tested: ISL-62 mg, ISL-98 mg, and ENG-33 mg. Animals received one or two ISL-eluting implants, with doses of 42, 66, or 108 µg of ISL/day with or without an additional ENG-33 mg implant (31 µg/day). Drug release increased linearly with dose with median [range] plasma ISL levels of 1.3 [1.0-2.5], 1.9 [1.2-6.3] and 2.8 [2.3-11.6], respectively. The ISL-62 and 98 mg implants demonstrated stable drug release over three months with ISL-triphosphate (ISL-TP) concentr54ations in PBMCs above levels predicted to be efficacious for PrEP. Similarly, ENG implants demonstrated sustained drug release with median [range] plasma ENG levels of 495 [229-1110] pg/mL, which suppressed progesterone within two weeks and showed no evidence of altering ISL pharmacokinetics. Two of the six ISL-98 mg implants broke during the study and induced implant-site reactions, whereas no reactions were observed with intact implants. We show that ISL and ENG biodegradable implants are safe and yield sufficient drug levels to achieve prevention targets. The evaluation of optimized implants with increased mechanical robustness is underway for improved durability and vaginal efficacy in a SHIV challenge model.

Unveiling targeted spatial metabolome of rice seed at the dough stage using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry imaging.

Rice (Oryza sativa) seeds contain a variety of metabolites, which not only provide energy for their own growth and development, but also are an important source of nutrition for humans. It is crucial to study the distribution of metabolites in rice seeds, but the spatial metabolome of rice seeds is rarely investigated. In this study, Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) imaging was used to reveal the spatial distribution of free soluble sugars (glucose, fructose, sucrose, and maltose), amino acids (9 essential amino acids and 2 amino acids affecting rice eating quality: L-aspartic acid and L-glutamic acid), and 4 metabolites in the flavonoids synthesis pathway (cinnamic acid, naringenin chalcone, naringenin, and dihydrokaempferol) in rice seed at the dough stage. It was found that the 4 free soluble sugars present similar spatial distribution, mainly distributed in the seed cortex and embryo with high abundance. The majority of amino acids are also concentrated in the rice cortex and embryo, while the others are abundant in the whole seed. Besides cinnamic acid distributed in the seed cortex and embryo, the naringenin chalcone, naringenin, and dihydrokaempferol were also found in the endosperm and had lower content. Furthermore, a colocalization phylogenetic tree according to the spatial distribution imaging of each metabolite was constructed. This study revealed the distribution diversity of metabolites in different segmentations of rice seed at the dough stage, providing clues for the nutritional differences between brown rice and white rice, and serving as a reference for people to target a healthy diet.

Corrigendum: Genome-wide characterization of NAC transcription factors in Camellia sinensis and the involvement of CsNAC28 in drought tolerance.

[This corrects the article DOI: 10.3389/fpls.2022.1065261.].

Corrigendum: Involvement of PtPHR1 in phosphates starvation-induced alkaloid biosynthesis in Pinellia ternata (Thunb.) Breit.

[This corrects the article DOI: 10.3389/fpls.2022.914648.].

Analysis of the Flavonoidome Reveals the Different Health-Promoting Flavonoid Characteristics in Fruit.

Flavonoids are one of the important metabolites of plants, and many flavonoids have functions of antioxidant or antimicrobial, which can help plants resist environmental stress. On the other hand, flavonoids also have a health-promoting effect for humans, such as antioxidant and anti-aging, and some flavonoids can assist in disease treatment. Fruit is one of the main sources of plant food and flavonoids intake for humans. Understanding the flavonoidome of various fruits is helpful to choose fruit combinations according to different demands. In this study, we explored the composition and relative content of flavonoids in 22 fruits and analyzed some health-promoting flavonoids in fruits. In addition, we selected several fruits and measured their antioxidant capacity through experiments. Our study initially established a database of fruit flavonoidome, and can provide reference for nutrition research, fruit breeding and industrial development.

Effect of allyl isothiocyanate on the growth and virulence of Clostridium perfringens and its application on cooked pork.

The objective of this study is to explore the antibacterial action modes and virulence-inhibitory effects of allyl isothiocyanate (AITC) against Clostridium perfringens (C. perfringens). The minimum inhibitory concentration (MIC) of AITC against vegetative cells of Cp 13124 was 0.1 µL/mL, and the time-kill kinetics analysis revealed that AITC could significantly suppress the growth of Cp 13124. According to the results from scanning electron microscopy (SEM), fluorescence microscopy, and UV absorbance substance detection, the cell membrane of Cp 13124 was damaged upon AITC treatment, causing a loss of integrity and the release of intracellular substances. Meanwhile, the fluorescence quenching experiment indicated the interaction of AIT-C with membrane proteins, which caused changes in the conformation of membrane proteins. Measurement of reactive oxygen species (ROS) and flow cytometry analysis demonstrated that AITC could induce apoptosis through oxidative stress. The formation of Cp 13124 biofilms was inhibited by AITC using the crystalline violet method, which was possibly related to the inhibition of sliding motility. Finally, low concentrations of AITC could be used as an antibacterial agent to inhibit the outgrowth of Cp 13124 in cooked pork, suggesting that AITC is a promising candidate for novel preservatives in the meat business.

BACE1 in PV interneuron tunes hippocampal CA1 local circuits and resets priming of fear memory extinction.

BACE1 is the rate-limiting enzyme for ß-amyloid (Aß) production and therefore is considered a prime drug target for treating Alzheimer's disease (AD). Nevertheless, the BACE1 inhibitors failed in clinical trials, even exhibiting cognitive worsening, implying that BACE1 may function in regulating cognition-relevant neural circuits. Here, we found that parvalbumin-positive inhibitory interneurons (PV INs) in hippocampal CA1 express BACE1 at a high level. We designed and developed a mouse strain with conditional knockout of BACE1 in PV neurons. The CA1 fast-spiking PV INs with BACE1 deletion exhibited an enhanced response of postsynaptic N-methyl-D-aspartate (NMDA) receptors to local stimulation on CA1 oriens, with average intrinsic electrical properties and fidelity in synaptic integration. Intriguingly, the BACE1 deletion reorganized the CA1 recurrent inhibitory motif assembled by the heterogeneous pyramidal neurons (PNs) and the adjacent fast-spiking PV INs from the superficial to the deep layer. Moreover, the conditional BACE1 deletion impaired the AMPARs-mediated excitatory transmission of deep CA1 PNs. Further rescue experiments confirmed that these phenotypes require the enzymatic activity of BACE1. Above all, the BACE1 deletion resets the priming of the fear memory extinction. Our findings suggest a neuron-specific working model of BACE1 in regulating learning and memory circuits. The study may provide a potential path of targeting BACE1 and NMDAR together to circumvent cognitive worsening due to a single application of BACE1 inhibitor in AD patients.

The CsHSFA-CsJAZ6 module-mediated high temperature regulates flavonoid metabolism in Camellia sinensis.

High temperatures (HTs) seriously affect the yield and quality of tea. Catechins, derived from the flavonoid pathway, are characteristic compounds that contribute to the flavour of tea leaves. In this study, we first showed that the flavonoid content of tea leaves was significantly reduced under HT conditions via metabolic profiles; and then demonstrated that two transcription factors, CsHSFA1b and CsHSFA2 were activated by HT and negatively regulate flavonoid biosynthesis during HT treatment. Jasmonate (JA), a defensive hormone, plays a key role in plant adaption to environmental stress. However, little has been reported on its involvement in HT response in tea. Herein, we demonstrated that CsHSFA1b and CsHSFA2 activate CsJAZ6 expression through directly binding to heat shock elements in its promoter, and thereby repress the JA pathway. Most secondary metabolites are regulated by JA, including catechin in tea. Our study reported that CsJAZ6 directly interacts with CsEGL3 and CsTTG1 and thereby reduces catechin accumulation. From this, we proposed a CsHSFA-CsJAZ6-mediated HT regulation model of catechin biosynthesis. We also determined that negative regulation of the JA pathway by CsHSFAs and its homologues is conserved in Arabidopsis. These findings broaden the applicability of the regulation of JAZ by HSF transcription factors and further suggest the JA pathway as a valuable candidate for HT-resistant breeding and cultivation.

InferLoop: leveraging single-cell chromatin accessibility for the signal of chromatin loop.

Deciphering cell-type-specific 3D structures of chromatin is challenging. Here, we present InferLoop, a novel method for inferring the strength of chromatin interaction using single-cell chromatin accessibility data. The workflow of InferLoop is, first, to conduct signal enhancement by grouping nearby cells into bins, and then, for each bin, leverage accessibility signals for loop signals using a newly constructed metric that is similar to the perturbation of the Pearson correlation coefficient. In this study, we have described three application scenarios of InferLoop, including the inference of cell-type-specific loop signals, the prediction of gene expression levels and the interpretation of intergenic loci. The effectiveness and superiority of InferLoop over other methods in those three scenarios are rigorously validated by using the single-cell 3D genome structure data of human brain cortex and human blood, the single-cell multi-omics data of human blood and mouse brain cortex, and the intergenic loci in the GWAS Catalog database as well as the GTEx database, respectively. In addition, InferLoop can be applied to predict loop signals of individual spots using the spatial chromatin accessibility data of mouse embryo. InferLoop is available at https://github.com/jumphone/inferloop.

An adaptive absorption spectroscopy with adjustable moving window width for suppressing nonlinear effects in absorbance measurements.

Absorption spectroscopy based on Lambert-Beer law has been widely used in material structure analysis, research in chemical reaction kinetics, and exploration of various physicochemical reaction mechanisms. However, serious nonlinearity between absorbance and measured concentration can occur in actual measurements. The idea of moving window is first introduced into the field of spectral nonlinearity in the paper. Combining with the characteristic absorption spectra of the substances to be measured, we propose an adaptive absorption spectroscopy (A-AS) with adjustable moving window parameters to effectively suppress the nonlinear effects in absorbance measurements. The validity of this method is verified by taking the differential optical absorption spectroscopy to detect SO2 as an example. The 210-230 nm characteristic absorption band is traversed and divided by the moving window with adjustable parameters, and the estimated coefficient (k-value) of each band is calculated. On this basis, all k-values are initially and secondly screened to obtain the optimal kbest, and then the optimal concentration value is obtained by inversion. Compared with the broad-band method and narrow-band method, it shows excellent performance that the maximum error and standard deviation of A-AS is only 1.3% and 3.8 in the entire concentration range, suggesting good linearity and stability in both high and low concentration environments. Therefore, it is inferred that A-AS is universally adaptable and enables dynamic linear measurements over wide concentration range.

Comparative metabolomics of flavonoids in twenty vegetables reveal their nutritional diversity and potential health benefits.

Vegetables are rich in flavonoids and are widely consumed in our daily life. However, comprehensive information on flavonoids components in vegetable varieties and the distribution of flavonoids with health-promoting effects in different vegetables are rarely investigated. Here, we analyzed the constitution of flavonoids among 20 vegetables by widely-targeted metabolome analysis. A total of 403 flavonoids were detected and classified as flavonoid, flavonols, anthocyanins, isoflavones, flavonoid carbonoside, dihydroflavone, chalcones, flavanols, dihydroflavonol, tannin, proanthocyanidins, and other flavonoids. Interestingly, we found that the content and types of flavonoids in bean sprouts and hot pepper were relatively abundant, whereas those were lower in carrot, lettuce, and Zizania latifolia. Then, we characterized the representative flavonoids including flavonoid, flavonols, chalcones, and isoflavones, and related them to the health-promoting effects of vegetables. Finally, we examined the relevance of the flavonoids to antioxidant capacity. Both bean sprouts and hot pepper possessed higher antioxidant enzyme activity, which were responsible for their great antioxidant capacity. Our study established a database of major flavonoids components in vegetables and further provides a new hint for the selection and breeding of vegetables based on their health-promoting effects.

Voltage-dependent anion channel 2 (VDAC2) facilitates the accumulation of rice stripe virus in the vector Laodelphax striatellus.

Rice stripe virus (RSV) causes enormous losses in rice production and is transmitted by the small brown planthopper, Laodelphax striatellus, in a persistent-propagative manner. RSV accumulation within the gut lumen of the vector is indispensable for the successful transmission to rice and insects. In this study, we obtained a 1464 bp full-length cDNA of a voltage-dependent anion channel 2 from L. striatellus (LsVDAC2), which encodes a 283 amino acid protein. RSV infection increased the expression of LsVDAC2 in the midguts and ovaries of L. striatellus by 260% and 228%, respectively. Silencing of LsVDAC2 resulted in a 88% reduction of RSV loads at 24 h after RNAi, indicating that LsVDAC2 facilitates RSV accumulation in the vector. Yeast two-hybrid and GST pulldown assays demonstrated that LsVDAC2 interacted with RSV RNA-dependent RNA polymerase, RdRp. Furthermore, experiments in vivo and in vitro showed that LsVDAC2 induced the apoptotic response in RSV-infected insects and tissues. Silencing of LsVDAC2 via RNAi significantly reduced the expression of genes for apoptosis-related caspases 1a and 1c by 62% and 78%, respectively, in RSV-infected vectors. Whether LsVDAC2-induced RSV accumulation is related to RSV RdRp and LsVDAC2-induced cell apoptosis deserves further investigation.

Comprehensive Genomic Analysis of Trihelix Family in Tea Plant (Camellia sinensis) and Their Putative Roles in Osmotic Stress.

In plants, Trihelix transcription factors are responsible for regulating growth, development, and reaction to various abiotic stresses. However, their functions in tea plants are not yet fully understood. This study identified a total of 40 complete Trihelix genes in the tea plant genome, which are classified into five clades: GT-1 (5 genes), GT-2 (8 genes), GTγ (2 genes), SH4 (7 genes), and SIP1 (18 genes). The same subfamily exhibits similar gene structures and functional domains. Chromosomal mapping analysis revealed that chromosome 2 has the most significant number of trihelix family members. Promoter analysis identified cis-acting elements in C. sinensis trihelix (CsTH), indicating their potential to respond to various phytohormones and stresses. The expression analysis of eight representative CsTH genes from four subfamilies showed that all CsTHs were expressed in more tissues, and three CsTHs were significantly induced under ABA, NaCl, and drought stress. This suggests that CsTHs plays an essential role in tea plant growth, development, and response to osmotic stress. Furthermore, yeast strains have preliminarily proven that CsTH28, CsTH36, and CsTH39 can confer salt and drought tolerance. Our study provides insights into the phylogenetic relationships and functions of the trihelix transcription factors in tea plants. It also presents new candidate genes for stress-tolerance breeding.

Genome-wide characterization of NAC transcription factors in Camellia sinensis and the involvement of CsNAC28 in drought tolerance.

The NAM, ATAF1/2, and CUC2 (NAC) transcription factors, which are members of a plant-specific gene family, play critical roles during the growth and development of plants and in their adaption to environmental stress. Few NAC transcription factors have been functionally characterized in tea plants (Camellia sinensis). Based on the analysis of the gene structure, motif pattern, and evolutionary relationship, we identified 104 NAC genes in C. sinensis. Among them, CsNAC28 is constitutively expressed in all organs, and most significantly, exhibiting remarkable responsiveness to abscisic acid (ABA) treatment and drought stress. ABA is a primary stress-related hormone. Recently, ABA-responsive element binding factor 2 (CsABF2) was identified in the ABA pathway of C. sinensis. However, the involvement of the CsABF2-mediated ABA pathway in regulating CsNACs was not known. Herein, a series of biochemical and genetic approaches supported the fact that CsNAC28 could potentially act as a transcription factor in the downstream of CsABF2. Furthermore, we investigated the function of CsNAC28 in the adapting of a plant to drought stress. The results showed that overexpression of CsNAC28 in Arabidopsis conferred hypersensitivity to ABA treatment and decreased the accumulation of reactive oxygen species (ROS), resulting in improved dehydration tolerance. Under conditions of drought, the expression levels of ABA pathway-related genes and drought stress‒inducible genes were greater in CsNAC28 overexpression lines than in the wild type. Our study's comprehensive characterization of NAC genes in C. sinensis could serve as a foundation for exploring the molecular mechanism of CsNAC-mediated drought responsiveness.