Identification of One O-Methyltransferase Gene Involved in Methylated Flavonoid Biosynthesis Related to the UV-B Irradiation Response in Euphorbia lathyris.

Flavonoids are ubiquitous polyphenolic compounds that play a vital role in plants' defense response and medicinal efficacy. UV-B radiation is a vital environmental regulator governing flavonoid biosynthesis in plants. Many plants rapidly biosynthesize flavonoids as a response to UV-B stress conditions. Here, we investigated the effects of flavonoid biosynthesis via UV-B irradiation in Euphorbia lathyris. We found that exposure of the E. lathyris callus to UV-B radiation sharply increased the level of one O-methyltransferase (ElOMT1) transcript and led to the biosynthesis of several methylated flavonoids. The methyltransferase ElOMT1 was expressed heterologously in E. coli, and we tested the catalytic activity of recombinant ElOMT1 with possible substrates, including caffeic acid, baicalin, and luteolin, in vitro. ElOMT1 could efficiently methylate when the hydroxyl groups were contained in the core nucleus of the flavonoid. This molecular characterization identifies a methyltransferase responsible for the chemical modification of the core flavonoid structure through methylation and helps reveal the mechanism of methylated flavonoid biosynthesis in Euphorbiaceae. This study identifies the O-methyltransferase that responds to UV-B irradiation and helps shed light on the mechanism of flavonoid biosynthesis in Euphorbia lathyris.

An Updated Review on Developing Small Molecule Kinase Inhibitors Using Computer-Aided Drug Design Approaches.

Small molecule kinase inhibitors (SMKIs) are of heightened interest in the field of drug research and development. There are 79 (as of July 2023) small molecule kinase inhibitors that have been approved by the FDA and hundreds of kinase inhibitor candidates in clinical trials that have shed light on the treatment of some major diseases. As an important strategy in drug design, computer-aided drug design (CADD) plays an indispensable role in the discovery of SMKIs. CADD methods such as docking, molecular dynamic, quantum mechanics/molecular mechanics, pharmacophore, virtual screening, and quantitative structure-activity relationship have been applied to the design and optimization of small molecule kinase inhibitors. In this review, we provide an overview of recent advances in CADD and SMKIs and the application of CADD in the discovery of SMKIs.

Evaluation of Inhibitory Effect and Mechanism of Euphorbia Factor L3 against Phytophthora capsici.

Phytophthora capsici is a highly destructive phytopathogenic oomycete with a broad host range and is responsible for tremendous losses. Euphorbia factor L3 (EFL3) is a natural plant-derived compound that has been widely studied in medicine and cosmetic applications. In this study, the sensitivity of 105 P. capsici isolates to EFL3 was determined, and the biological activity and physiological effects of EFL3 against P. capsici were investigated. The median effective concentration (EC50) values for EFL3 inhibition mycelial growth and spore germination ranged from 0.66 to 8.94 µg/mL (mean, 2.96 ± 0.91 µg/mL) and 1.63 to 13.16 µg/mL (mean, 5.30 ± 1.64 µg/mL), respectively. EFL3 treatment resulted in cell wall and cell membrane damage of P. capsici, which was revealed by morphological and ultrastructural observations, propidium iodide (PI) and calcofluor white (CFW) staining, and measurements of relative conductivity as well as malondialdehyde (MDA) and glycerol contents. In addition, the contents of phospholipid and cellulose, which are the major components of cell membrane and cell wall, were significantly reduced following EFL3 treatment. Furthermore, EFL3 provided protective as well as curative efficacies against P. capsici on detached tomato leaves and pepper seedlings in vivo. These data show that EFL3 exhibits strong inhibitory activity against P. capsici, thereby suggesting that it could be an effective alternative for controlling P. capsici-induced diseases.

Chemical Composition and Antifungal Activity of Zanthoxylum armatum Fruit Essential Oil against Phytophthora capsici.

Pathogenic plant oomycetes cause devastating damage to fruits and vegetables worldwide. Plant essential oils (EOs) are known to be promising candidates for the development of fungicides. In this study, we isolated twelve EOs from Tetradium ruticarpum, Tetradium daniellii, Tetradium fraxinifolium, Zanthoxylum armatum, Ruta graveolens, and Citrus medica leaves and fruits. We then investigated their chemical composition and antifungal activity against phytopathogenic oomycetes. Our results demonstrated that Z. armatum fruit essential oil (ZFO) in particular substantially inhibited the mycelial growth of Phytophthora capsici. Similarly, ZFO also strongly suppressed spore production and germination of P. capsici, and the application of ZFO significantly reduced disease symptoms caused by P. capsici in pepper. Furthermore, results from microscopic and biochemical studies indicated that ZFO damaged the ultrastructure and destroyed the membrane integrity of P. capsici, leading to the leakage of the cellular contents and ultimately causing cell death. It was concluded that ZFO could enhance the activities of defense-related enzymes in pepper fruits, which may also be responsible for the inhibition of phytophthora disease. Moreover, linalool and D-limonene were proven to be the primary effective components of ZFO. Our results collectively indicate that ZFO could be a potential candidate for the management of disease caused by P. capsici.

Inhibitory Effects of the Natural Product Esculetin on Phytophthora capsici and Its Possible Mechanism.

Esculetin is an important plant-derived natural product that has multiple bioactivities and applications. Phytophthora capsici is a notorious plant pathogen capable of infecting a broad range of hosts. In this study, we evaluated the antifungal activity of esculetin against P. capsici. The baseline sensitivity of P. capsici to esculetin was established using 108 isolates collected from various geographical regions in the Jiangsu and Shandong Provinces of China. The median effective concentration (EC50) values for esculetin ranged from 2.08 to 16.46 µg/ml (mean, 6.87 ± 2.70 µg/ml) and were normally distributed. Furthermore, both zoospore production and germination were strongly inhibited by esculetin. Importantly, esculetin exhibited protective as well as curative activities against P. capsici on tomato and was capable of restricting the early infection of P. capsici on Nicotiana benthamiana. We found that the esculetin treatment led to cell membrane damage of P. capsici, as revealed by morphological observations and measurements of relative conductivity and malondialdehyde (MDA). Finally, our results also suggested that esculetin may adversely affect P. capsici by inhibiting its DNA and protein synthesis. These findings will contribute to the broader evaluation of the use of esculetin to control diseases caused by P. capsici and toward a better understanding of its mode of action as a potential fungicide.

Genetic diversity, population structure and association analysis in cut chrysanthemum (Chrysanthemum morifolium Ramat.).

Characterizing the genetic diversity present in a working set of plant germplasm can contribute to its effective management and genetic improvement. The cut flower chrysanthemum (Chrysanthemum morifolium Ramat.) is an economically important ornamental species. With the repeated germplasm exchange and intensive breeding activities, it remains a major task in genetic research. The purpose of the present study was to characterize the genetic diversity and the population structure of a worldwide collection of 159 varieties, and to apply an association mapping approach to identify DNA-based markers linked to five plant architecture traits and six inflorescence traits. The genotyping demonstrated that there was no lack of genetic diversity in the collection and that pair-wise kinship values were relatively low. The clustering based on a Bayesian model of population structure did not reflect known variation in either provenance or inflorescence type. A principal coordinate analysis was, however, able to discriminate most of the varieties according to both of these criteria. About 1 in 100 marker pairs exhibited a degree of linkage disequilibrium. The association analysis identified a number of markers putatively linked to one or more of the traits. Some of these associations were robust over two seasons. The findings provide an in-depth understanding of genetic diversity and population structure present in cut flower chrysanthemum varieties, and an insight into the genetic control of plant architecture and inflorescence-related traits.

Genetic variation and association mapping of waterlogging tolerance in chrysanthemum.

MAIN CONCLUSION: Forty-five molecular markers were detected significantly associated with chrysanthemum' waterlogging tolerance, and four favorable parental lines were identified as potential donors for improving waterlogging tolerance in chrysanthemum. The productivity of chrysanthemum is downgraded by waterlogging soils, which has driven a search for germplasm showing an enhanced level of waterlogging tolerance (WT). As yet little is known regarding the mode of inheritance of WT in chrysanthemum. The study set out to characterize the extent of genetic variation for WT represented in a collection of one hundred chrysanthemum accessions by testing them under both greenhouse and field conditions. A membership function value of waterlogging (MFVW), which integrated a wilting index, a chlorosis score and the proportion of dead leaf in waterlogged plants, was used as a measure of WT. The variation for MFVW among plants grown in the greenhouse (two experiments) was generally higher than that generated in field-grown (one experiment) plants. The MFVW broad sense heritability was 0.82, and the phenotypic coefficient of variation (31.8 %) was larger than the genetic one (28.8 %). Association mapping (AM) identified 45 markers related to WT: 25 by applying the general linear model (GLM) + principal component (PC) model, 16 by applying the mixed linear model (MLM), 31 by applying the MLM + Q matrix model and 12 by applying the MLM + PC model. Of the associated markers, eight and two were predictive in two and three experiments within all models, respectively; the proportion of the phenotypic variance explained by the eight associations ranged from 6.3 to 16.4 %. On the basis of their harboring all four of the leading markers E2M16-2, SSR150-6, E19M16-1 and E10M10-12, the varieties 'Nannong Xuefeng', 'Qx097', 'Nannong Xunzhang' and 'Finch' were identified as potential donors for future improvement of WT in chrysanthemum.

Antifungal activity of the botanical compound rhein against Phytophthora capsici and the underlying mechanisms.

BACKGROUND: Phytophthora capsici is an extremely destructive phytopathogenic oomycete that causes huge economic losses. However, due to the drug resistance risk and environmental threat of chemical fungicides, it is necessary to develop environmentally friendly biocontrol alternatives. Rhein is a major medicinal ingredient of traditional Chinese herbs, and it is widely used in the medical field. However, its inhibitory effect against phytopathogens is unknown. Herein, the antifungal spectrum of rhein and its possible action mechanism against P. capsici were investigated. RESULTS: Rhein possessed broad-spectrum antifungal activity against phytopathogens, particularly P. capsici, Phytophthora infestans, Helminthosporium maydis, and Rhizoctonia solani. Rhein inhibited the mycelial growth as well as the spore germination of P. capsici with mean 50% effective concentration (EC50 ) values of 4.68 µg mL-1 and 6.57 µg mL-1 against 117 P. capsici isolates, respectively. Rhein effectively suppressed the occurrence and spread of Phytophthora blight and significantly destroyed the cell membrane permeability and integrity of P. capsici, corroded its cell wall integrity, and damaged its morphology and ultrastructure. Moreover, rhein caused a considerable reduction in the phospholipid and cellulose contents. Genome-wide transcriptional profiling of P. capsici in response to rhein indicated significant reduction in the expression levels of genes participating in glycerolipid metabolism and starch and sucrose metabolism. Additionally, rhein strengthened the disease defense system of pepper by enhancing related enzyme activities. CONCLUSION: This study demonstrated that rhein could effectively inhibit P. capsici using multiple mechanisms of action. Rhein has the potential to be an efficient alternative to control diseases caused by P. capsici. © 2023 Society of Chemical Industry.

Targeted discovery of unusual diterpenoids with anti-fungal activity from the root of Euphorbia lathyris.

Lathyrisone A (1), a diterpene with an undescribed tricyclic 6/6/6 fused carbon skeleton, along with spirolathyrisins B-D (3-5), three diterpenes with a rare [4.5.0] spirocyclic carbon skeleton, and one known compound (2) were isolated from the roots of Euphorbia lathyris. Their chemical structures were characterized by extensive spectroscopic analysis, X-ray crystallography, ECD and quantum chemistry calculation. A plausible biosynthetic pathway for compounds 1-5 was proposed, which suggested it is a competitive pathway for ingenol biosynthesis in the plant. The anti-fungal activities of these compounds were tested, especially, compound 2 showed stronger anti-fungal activities against Fusarium oxysporum and Alternaria alternata than the positive control fungicide thiophanate-methyl. The preliminary structure-activity relationship of compounds 1-5 was also discussed. These results not only expanded the chemical diversities of E. lathyris, but also provided a lead compound for the control of plant pathogens.

Identification and Functional Characterization of a Novel Sinapyl Alcohol Acyltransferase from Euphorbia lathyris L.

Methoxyeugenol is a phenylpropene compound derived from plants and has various bioactivities. The chemical synthesis of methoxyeugenol is accompanied by pollution issues, whereas extraction from plants is associated with problems such as low yield and high cost. The production of methoxyeugenol can be effectively addressed through an enzymatic approach. In this study, the acyltransferase genes of Euphorbia lathyris L. were screened by homologous alignment of the transcriptome data of E. lathyris in the late growth stage and the acyltransferase genes of the closely related plant species. The results showed that ElBAHD10 had the closest relationship with earlier reported ScCFAT and PhCFAT, which were found to catalyze the reaction of coniferyl alcohol to generate coniferyl acetate. The ElBAHD10 gene was successfully cloned from E. lathyris and subsequently expressed in Escherichia coli. The purified protein ElBAHD10 catalyzed the reaction of sinapyl alcohol with acetyl CoA and cinnamoyl CoA to form sinapyl acetate and sinapyl cinnamate, respectively. In contrast, the crude ElBAHD10 protein could catalyze sinapyl alcohol to directly generate methoxyeugenol. The recombinant E. coli strain expressing ElBAHD10 produced methoxyeugenol through whole-cell transformation. This study provides insights and lays the foundation for methoxyeugenol production through biosynthetic approaches.

Two lathyrane diterpenoid stereoisomers containing an unusual trans-gem-dimethylcyclopropane from the seeds of Euphorbia lathyris.

Two novel lathyrane-type diterpenoids, the Euphorbia factors L2a (1) and L2b (2), and their stereoisomer Euphorbia factor L2 (3) were obtained from seeds of Euphorbia lathyris. Both Euphorbia factors L2a and L2b possess an unprecedented trans-gem-dimethylcyclopropane as structural feature. Also, the Euphorbia factor L2a is the first example of a lathyrane diterpenoid with an endocyclic 12(Z)-double bond. The structures of the molecules and their absolute configurations were elucidated by comprehensive spectroscopic analyses, Cu-Kα radiation X-ray diffraction, and comparison with calculated electronic circular dichroism (ECD) data. The Euphorbia factor L2b exhibited an inhibitory effect against U937 cell line with an IC50 value of 0.87 µM.

The CmTCP20 gene regulates petal elongation growth in Chrysanthemum morifolium.

Chrysanthemum morifolium is one of the most popular ornamental species worldwide, with high ornamental and economic value. Petal size is an important factor that influences the ornamental value. CmTCP20 is a member of TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTORs (TCPs) gene family, which is closely associated with the growth and development of plants. Our previous study found that the expression of CmTCP20 was obviously down-regulated during chrysanthemum petal elongation, but its function in petal elongation has not yet been revealed. We show here that the overexpression CmTCP20 in Arabidopsis and chrysanthemum leads to similar phenotypes, including larger flower buds (or inflorescences) and longer petals. Interestingly, ectopic expression in Schizosaccharomyces pombe yeast cells showed that CmTCP20 could repress cell division and promote cell elongation. Moreover, the yeast two-hybrid, BiFC and pull-down experimental results indicated that CmTCP20 may regulate petal size via interacting with CmJAZ1-like and inducing down-regulation of CmBPE2 gene expression. This study preliminarily clarifies the function of CmTCP20 on chrysanthemum petal elongation, providing the basic theory for improving the ornamental characteristic of chrysanthemum.

Association analysis of drought tolerance in cut chrysanthemum (Chrysanthemum morifolium Ramat.) at seedling stage.

Understanding the genetic architecture is a prerequisite for crop improvement. The current research aimed to characterize the extent of genetic variation of drought tolerance harbored in a global collection of 159 chrysanthemum cultivars over 2 years. An average subordinate function value (ASFV), integrating the wilting index, the fresh weight retention rate, and the survival rate after re-watering under two drought-stressed trials, was used to quantify the level of drought tolerance. The performance of ASFV was generally correlated between the 2 years; and a high magnitude (0.95) of broad-sense heritability, coupled with the moderate genetic advance, was estimated for the ASFV. By applying MLM model with both population structure and kinship matrix as covariates association mapping identified 16 markers linked to drought tolerance, with the proportion of the phenotypic variation explained by an individual marker ranging from 4.4 to 7.6%. Of the eight markers predictive across the 2 years, four (E11M24-9, E3M2-8, E1M5-5, and EST-SSR34-3) were identified as favorable alleles for drought tolerance. Several cultivars that carry at least three of the four favorable alleles were identified as potential donor parents for future improvement of the drought tolerance. The findings provide an insight into the genetic basis of the drought tolerance in chrysanthemum and will, therefore, aid in developing new cultivars with enhanced tolerance against drought stress.

Next-generation sequencing of the Chrysanthemum nankingense (Asteraceae) transcriptome permits large-scale unigene assembly and SSR marker discovery.

BACKGROUND: Simple sequence repeats (SSRs) are ubiquitous in eukaryotic genomes. Chrysanthemum is one of the largest genera in the Asteraceae family. Only few Chrysanthemum expressed sequence tag (EST) sequences have been acquired to date, so the number of available EST-SSR markers is very low. METHODOLOGY/PRINCIPAL FINDINGS: Illumina paired-end sequencing technology produced over 53 million sequencing reads from C. nankingense mRNA. The subsequent de novo assembly yielded 70,895 unigenes, of which 45,789 (64.59%) unigenes showed similarity to the sequences in NCBI database. Out of 45,789 sequences, 107 have hits to the Chrysanthemum Nr protein database; 679 and 277 sequences have hits to the database of Helianthus and Lactuca species, respectively. MISA software identified a large number of putative EST-SSRs, allowing 1,788 primer pairs to be designed from the de novo transcriptome sequence and a further 363 from archival EST sequence. Among 100 primer pairs randomly chosen, 81 markers have amplicons and 20 are polymorphic for genotypes analysis in Chrysanthemum. The results showed that most (but not all) of the assays were transferable across species and that they exposed a significant amount of allelic diversity. CONCLUSIONS/SIGNIFICANCE: SSR markers acquired by transcriptome sequencing are potentially useful for marker-assisted breeding and genetic analysis in the genus Chrysanthemum and its related genera.

Functional characterization of a Chrysanthemum dichrum stress-related promoter.

A 1,474-bp stress-inducible CdDREBa promoter was identified from Chrysanthemum dichrum, revealing several candidate stress-related cis-acting elements (MYC-box, MYB site, GT-1, and W-box) within it. In Arabidopsis leaf tissues transformed with a CdDREBa promoter-ß-glucuronidase (GUS) gene fusion, serially 5'-deleted CdDREBa promoters were differentially activated by cold and salinity. Histochemical and quantitative assays of GUS expression allowed us to localize a critical part of the promoter located between upstream 430 and 351 nt. This 80-bp fragment enhanced GUS expression under salinity stress when fused to -90/+8 CaMV 35S minimal promoter. Further promoter internal-deletion assays indicated that a low temperature-responsive element was located between positions -430 and -390, and a salinity inducible one between -385 and -351. Our results showed that there was a novel stress-related critical region except for the known cis-acting element (MYC-box, GT-1) in CdDREBa promoter.