Systematic identification and functional characterization of the CFEM proteins in fishscale bamboo rhombic-spot pathogen Neostagonosporella sichuanensis.

Fungal effectors play a crucial role in the interaction between pathogenic fungi and their hosts. These interactions directly influence the invasion and spread of pathogens, and the development of diseases. Common in fungal extracellular membrane (CFEM) effectors are closely associated with the pathogenicity, cell wall stability, and pathogenic processes of pathogenic fungi. The aim of this study was to investigate the role of CFEM proteins in Neostagonosporella sichuanensis in pathogen-host interactions. We retrieved 19 proteins containing CFEM structural domains from the genome of N. sichuanensis. By systematic analysis, five NsCFEM proteins had signal peptides but lacked transmembrane structural domains, and thus were considered as potential effectors. Among them, NsCFEM1 and NsCFEM2 were successfully cloned and their functions were further investigated. The validation results show that NsCFEM1 was localized in the cell membrane and nucleus, whereas NsCFEM2 was exclusively observed in the cell membrane. Both were identified as secreted proteins. Additionally, NsCFEM1 inhibited Bax-induced programmed cell death in Nicotiana benthamiana, whereas NsCFEM2 did not induce or inhibit this response. NsCFEM1 was implicated as a virulence factor that contributes to fungal growth, development, stress response, and pathogenicity. NsCFEM2 was implicated in maintenance of cell wall stability. This study lays a foundation for elucidating the role of CFEM proteins in the pathogen of fishscale bamboo rhombic-spot caused by N. sichuanensis. In particular, the functional studies of NsCFEM1 and NsCFEM2 revealed their potential roles in the interaction between N. sichuanensis and the host Phyllostachys heteroclada.

Molecular and Functional Characterization of Pheromone Binding Protein 2 from Cyrtotrachelus buqueti (Coleoptera: Curculionidae).

Pheromone-binding proteins (PBPs) play important roles in binding and transporting sex pheromones. However, the PBP genes identified in coleopteran insects and their information sensing mechanism are largely unknown. Cyrtotrachelus buqueti (Coleoptera: Curculionidae) is a major insect pest of bamboo plantations. In this study, a novel PBP gene, CbuqPBP2, from C. buqueti was functionally characterized. CbuqPBP2 was more abundantly expressed in the antennae of both sexes than other body parts, and its expression level was significantly male-biased. Fluorescence competitive binding assays showed that CbuqPBP2 exhibited the strongest binding affinity to dibutyl phthalate (Ki = 6.32 µM), followed by styrene (Ki = 11.37 µM), among twelve C. buqueti volatiles. CbuqPBP2, on the other hand, showed high binding affinity to linalool (Ki = 10.55), the main volatile of host plant Neosinocalamus affinis. Furthermore, molecular docking also demonstrated the strong binding ability of CbuqPBP2 to dibutyl phthalate, styrene, and linalool, with binding energy values of -5.7, -6.6, and -6.0 kcal/mol, respectively, and hydrophobic interactions were the prevailing forces. The knockdown of CbuqPBP2 expression via RNA interference significantly reduced the electroantennography (EAG) responses of male adults to dibutyl phthalate and styrene. In conclusion, these results will be conducive to understanding the olfactory mechanisms of C. buqueti and promoting the development of novel strategies for controlling this insect pest.

Uncovering the mechanisms of salicylic acid-mediated abiotic stress tolerance in horticultural crops.

Salicylic acid (SA) has been recognized as a promising molecule for improving abiotic stress tolerance in plants due to its ability to enhance antioxidant defense system, and promote root architecture system. Recent research has focused on uncovering the mechanisms by which SA confers abiotic stress tolerance in horticultural crops. SA has been shown to act as a signaling molecule that triggers various physiological and morphological responses in plants. SA regulates the production of reactive oxygen species (ROS). Moreover, it can also act as signaling molecule that regulate the expression of stress-responsive genes. SA can directly interact with various hormones, proteins and enzymes involved in abiotic stress tolerance. SA regulates the antioxidant enzymes activities that scavenge toxic ROS, thereby reducing oxidative damage in plants. SA can also activate protein kinases that phosphorylate and activate transcription factors involved in stress responses. Understanding these mechanisms is essential for developing effective strategies to improve crop resilience in the face of changing environmental conditions. Current information provides valuable insights for farmers and plant researchers, offering new strategies to enhance crop resilience and productivity in the face of environmental challenges. By harnessing the power of SA and its signaling pathways, farmers can develop more effective stress management techniques and optimize crop performance. Plant researchers can also explore innovative approaches to breed or engineer crops with enhanced stress tolerance, thereby contributing to sustainable agriculture and food security.

CRISPR/Cas9 mutated p-coumaroyl shikimate 3'-hydroxylase 3 gene in Populus tomentosa reveals lignin functioning on supporting tree upright.

The lignin plays one of the most important roles in plant secondary metabolism. However, it is still unclear how lignin can contribute to the impressive height of wood growth. In this study, C3'H, a rate-limiting enzyme of the lignin pathway, was used as the target gene. C3'H3 was knocked out by CRISPR/Cas9 in Populus tomentosa. Compared with wild-type popular trees, c3'h3 mutants exhibited dwarf phenotypes, collapsed xylem vessels, weakened phloem thickening, decreased hydraulic conductivity and photosynthetic efficiency, and reduced auxin content, except for reduced total lignin content and significantly increased H-subunit lignin. In the c3'h3 mutant, the flavonoid biosynthesis genes CHS, CHI, F3H, DFR, ANR, and LAR were upregulated, and flavonoid metabolite accumulations were detected, indicating that decreasing the lignin biosynthesis pathway enhanced flavonoid metabolic flux. Furthermore, flavonoid metabolites, such as naringenin and hesperetin, were largely increased, while higher hesperetin content suppressed plant cell division. Thus, studying the c3'h3 mutant allows us to deduce that lignin deficiency suppresses tree growth and leads to the dwarf phenotype due to collapsed xylem and thickened phloem, limiting material exchanges and transport.

Ethylene-induced improvement in photosynthetic performance of Zanthoxylum armatum under reoxygenation conditions.

In this study, we evaluated the photosynthetic performance of Zanthoxylum armatum seedlings to test the tolerance to reoxygenation after waterlogging. The experiment included a control group without waterlogging (NW) and three reoxygenation groups with reoxygenation after 1day (WR1), 2days (WR2) and 3days (WR3). Seedlings were pretreated with concentrations of 0, 200 and 400µmolL-1 of ethylene. The results showed that reoxygenation after waterlogging for 1-3days decreased photosynthetic pigments content, enzymes activity, stomatal conductance (G s ), net photosynthetic rate (P n ), transpiration rate (T r ) and water-use efficiency (WUE). However, pretreatment with ethylene increased photosynthetic pigments content, enzymes activity and gas exchange parameters under both NW and WR3 treatments. The chlorophyll fluorescence results showed that the maximum quantum yield of PSII (F v /F m ) and actual photochemical efficiency of PSII (Φ PSII ) remained no significant changes under the NW and WR1 treatments, while they were significantly reduced with an increase in waterlogging days followed by reoxygenation under WR2 and WR3 treatments. Exogenous ethylene inhibited F v /F m and the non-photochemical quenching coefficient (NPQ), while enhanced Φ PSII and electron transfer efficiency (ETR) under WR2 treatments. Moreover, the accumulation of exogenous ethylene reduced photosynthetic ability. These findings provide insights into the role of ethylene in enhancing the tolerance of Z. armatum to reoxygenation stress, which could help mitigate the impact of continued climate change.

Genome-wide characterization of the MBF1 gene family and its expression pattern in different tissues and stresses in Zanthoxylum armatum.

BACKGROUND: Multiprotein bridging factor 1 (MBF1) is a crucial transcriptional coactivator in animals, plants, and some microorganisms, that plays a necessary role in growth development and stress tolerance. Zanthoxylum armatum is an important perennial plant for the condiments and pharmaceutical industries, whereas the potential information in the genes related to stress resistance remains poorly understood in Z. armatum.  RESULTS: Herein, six representative species were selected for use in a genome-wide investigation of the MBF1 family, including Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Citrus sinensis, Ginkgo biloba, and Z. armatum. The results showed that the MBF1 genes could be divided into two groups: Group I contained the MBF1a and MBF1b subfamilies, and group II was independent of the MBF1c subfamily.. Most species have at least two different MBF1 genes, and MBF1c is usually an essential member. The three ZaMBF1 genes were respectively located on ZaChr26, ZaChr32, and ZaChr4 of Zanthoxylum chromosomes. The collinearity were occurred between three ZaMBF1 genes, and ZaMBF1c showed the collinearity between Z. armatum and both P. trichocarpa and C. sinensis. Moreover, many cis-elements associated with abiotic stress and phytohormone pathways were detected in the promoter regions of MBF1 of six representative species. The ERF binding sites were the most abundant targets in the sequences of the ZaMBF1 family, and some transcription factor sites related to floral differentiation were also identified in ZaMBF1c, such as MADS, LFY, Dof, and AP2. ZaMBF1a was observed to be very highly expressed in 25 different samples except in the seeds, and ZaMBF1c may be associated with the male and female floral initiation processes. In addition, expression in all the ZaMBF1 genes could be significantly induced by water-logging, cold stress, ethephon, methyl jasmonate, and salicylic acid treatments, especially in ZaMBF1c. CONCLUSION: The present study carried out a comprehensive bioinformatic investigation related to the MBF1 family in six representative species, and the responsiveness of ZaMBF1 genes to various abiotic stresses and phytohormone inductions was also revealed. This work not only lays a solid foundation to uncover the biological roles of the ZaMBF1 family in Z. armatum, but also provides some broad references for conducting the MBF1 research in other plants.

Integrated transcriptome and plant growth substance profiles to identify the regulatory factors involved in floral sex differentiation in Zanthoxylum armatum DC.

Zanthoxylum armatum is a prominent plant for food industries. Its male flowers often occur in gynogenesis plants; however, the potential mechanism remains poorly understood. Herein, a total of 26 floral sex differentiation stages were observed to select four vital phases to reveal key factors by using RNA-seq, phytohormones and carbohydrates investigation. The results showed that a selective abortion of stamen or pistil primordia could result in the floral sex differentiation in Z. armatum. Carbohydrates might collaborate with cytokinin to effect the male floral differentiation, whereas female floral differentiation was involved in SA, GA1, and ABA biosynthesis and signal transduction pathways. Meanwhile, these endogenous regulators associated with reproductive growth might be integrated into ABCDE model to regulate the floral organ differentiation in Z. armatum. Furthermore, the 21 crucial candidates were identified in co-expression network, which would contribute to uncovering their roles in floral sex differentiation of Z. armatum in further studies. To the best of our knowledge, this study was the first comprehensive investigation to link floral sex differentiation with multi-level endogenous regulatory factors in Z. armatum. It also provided new insights to explore the regulatory mechanism of floral sex differentiation, which would be benefited to cultivate high-yield varieties in Z. armatum.

Conversion effects of farmland to Zanthoxylum bungeanum plantations on soil organic carbon mineralization in the arid valley of the upper reaches of Yangtze River, China.

Farmland conversion to forest is considered to be one of the effective measures to mitigate climate change. However, the impact of farmland conversion to forest land or grassland on soil CO2 emission in arid areas is unclear due to the lack of comparative information on soil organic carbon (SOC) mineralization of different conversion types. The SOC mineralization in 0-100 cm soil layer in farmland (FL), abandoned land (AL) and different ages (including 8, 15, 20 and 28 years) of Zanthoxylum bungeanum plantations were measured by laboratory incubation. The size and decomposition rate of fast pool (Cf) and slow pool (Cs) in different land-use types and soil layers were estimated by double exponential model. The results showed that: 1) Farmland conversion increased the cumulative CO2-C release (Cmin) and SOC mineralization efficiency, and those indexes in AL were higher than that in Z. bungeanum plantations. The Cmin and SOC mineralization efficiency of 0-100 cm soil increased with the ages of Z. bungeanum plantation. Both Cmin and SOC mineralization efficiency decreased with the increase of soil depth; 2) Both soil Cf and Cs increased after farmland converted to Z. bungeanum plantations and AL. The Cs in the same soil layer increased with the ages of Z. bungeanum plantation, and the Cf showed a "V" type with the increased ages of Z. bungeanum plantation. The Cf and Cs decreased with the increase of soil depth in all land-use types; 3) Farmland conversion increased the decomposition rate of Cf (k1) in all soil layer by 0.008-0.143 d-1 and 0.082-0.148 d-1 in Z. bungeanum plantations and AL, respectively. The k1 was obviously higher in the 0-20 cm soil layer than that in other soil layers, while the decomposition rate of Cs (k2) was not affected by FL conversion and soil depth; and 4) The initial soil chemical properties and enzyme activity affected SOC mineralization, especially the concentrations of total organic nitrogen (TON), SOC, easily oxidizable organic carbon (EOC) and microbial biomass carbon (MBC). It indicated that the conversion of farmland to Z. bungeanum plantations and AL increases SOC mineralization, especially in deeper soils, and it increased with the ages. The conversion of farmland to Z. bungeanum plantation is the optimal measure when the potential C sequestration of plant-soil system were taken in consideration.

Ectopic expression of an AGAMOUS homologue gene in Jatropha curcas causes early flowering and heterostylous phenotypes.

Jatropha curcasseeds are abundant in biodiesel, and low seed yields are linked to poor quality female flowers, which creates a bottleneck for Jatropha seed utilization. Therefore, identifying the genes associated with flowering is crucial for the genetic enrichment of seed yields. Here, we identified an AGAMOUS homologue gene (JcAG) from J. curcas. We found that reproductive organs had higher JcAG expression than vegetative organs, particularly the carpel. Rosette leaves were small and misshapen in 35S:JcAG transgenic lines in comparison with those in wild-type plants. JcAG overexpression caused an extremely early flowering, delayed perianth and stamen filament development, small flowers, and significantly shorter Arabidopsis plants with little fruit. In the JcAG-overexpressing line, the homeotic transformation of sepals into pistillate organs was observed, and floral meristem and organ identity genes were regulated. This study provides insights into the JcAG's function and benefits to our knowledge of the underlying the genetic mechanisms related to floral sex differentiation in Jatropha.

De novo transcriptome assembly for the five major organs of Zanthoxylum armatum and the identification of genes involved in terpenoid compound and fatty acid metabolism.

BACKGROUND: Zanthoxylum armatum (Z. armatum) is a highly economically important tree that presents a special numbing taste. However, the underlying regulatory mechanism of the numbing taste remains poorly understood. Thus, the elucidation of the key genes associated with numbing taste biosynthesis pathways is critical for providing genetic information on Z. armatumand the breeding of high-quality germplasms of this species. RESULTS: Here, de novo transcriptome assembly was performed for the five major organs of Z. armatum, including the roots, stems, leaf buds, mature leaves and fruits. A total of 111,318 unigenes were generated with an average length of 1014 bp. Additionally, a large number of SSRs were obtained to improve our understanding of the phylogeny and genetics of Z. armatum. The organ-specific unigenes of the five major samples were screened and annotated via GO and KEGG enrichment analysis. A total of 53 and 34 unigenes that were exclusively upregulated in fruit samples were identified as candidate unigenes for terpenoid biosynthesis or fatty acid biosynthesis, elongation and degradation pathways, respectively. Moreover, 40 days after fertilization (Fr4 stage) could be an important period for the accumulation of terpenoid compounds during the fruit development and maturation of Z. armatum. The Fr4 stage could be a key point at which the first few steps of the fatty acid biosynthesis process are promoted, and the catalysis of subsequent reactions could be significantly induced at 62 days after fertilization (Fr6 stage). CONCLUSIONS: The present study realized de novo transcriptome assembly for the five major organs of Z. armatum. To the best of our knowledge, this study provides the first comprehensive analysis revealing the genes underlying the special numbing taste of Z. armatum. The assembled transcriptome profiles expand the available genetic information on this species and will contribute to gene functional studies, which will aid in the engineering of high-quality cultivars of Z. armatum.

[Simultaneous determination of leucovorin and 5-methyl-tetrahydrofolate diastereoisomers in human plasma by high-performance liquid chromatography-tandem mass spectrometry coupled with ultrafiltration centrifugation-based pretreatment and its application to a pharmacokinetic study].

A simple, sensitive, and stable high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for the simultaneous determination of leucovorin and 5-methyltetrahydrofolate diastereomers in human plasma using methotrexate as the internal standard. The analytes and the internal standard were extracted from plasma samples by simple ultrafiltration centrifugation-based extraction. The separation was achieved on a chiral HSA column (150 mm×4 mm, 5 µm) using mobile phases containing 10 mmol pH 8.0 ammonium acetate and acetonitrile in gradient mode. The method showed good linearities in the ranges of 25-5000 µg/L and 12.5-3000 µg/L for leucovorin and 5-methyltetrahydrofolate diastereoisomers, respectively. The method was fully validated with respect to sensitivity, precision, accuracy, matrix effect, extraction recovery, and stability of analytes under various conditions. The method was successfully applied to a pharmacokinetic study of 125 mg/m2 6R,S-leucovorin and 62.5 mg/m2 6S-leucovorin. The results showed that the maximum observed concentrations (Cmax) of 6S-leucovorin and L-5-methyltetrahydrofolate were (3137.917±408.837) and (1679.633±244.132) µg/L, respectively, and the areas under the curve from the time of dosing to the last measurable concentration (AUC0-t) were (7504.883±1185.101) and (14001.214±2868.949) µg/L in the 125 mg/m2 6R,S-leucovorin dose group. The Cmax values of 6S-leucovorin and L-5-methyltetrahydrofolate were (3187.917±387.298) and (1739.204±224.755) µg/L, respectively, and AUC0-t values were (7426.664±854.825) and (14884.331±1843.353) µg/L in the 62.5 mg/m2 6S-leucovorin dose group. There were no significant diffe-rences in the main pharmacokinetic parameters between the two dose groups, and the pharmacokinetic characteristics as well as the rate and extent of absorption were consistent. This method can provide technical support for future bioequivalence studies of sodium leucovorin.

Analysis of Transcriptional Responses of the Inflorescence Meristems in Jatropha curcas Following Gibberellin Treatment.

Jatropha curcas L. seeds an oilseed plant with great potential for biodiesel production. However, low seed yield, which was limited by its lower female flowers, was a major drawback for its utilization. Our previous study found that the flower number and female-to-male ratio were increased by gibberellin treatment. Here, we compared the transcriptomic profiles of inflorescence meristem at different time points after gibberellic acid A3 (GA3) treatment. The present study showed that 951 differentially expressed genes were obtained in response to gibberellin treatment, compared with control samples. The 6-h time point was an important phase in the response to exogenous gibberellin. Furthermore, the plant endogenous gibberellin, auxin, ethylene, abscisic acid, and brassinolide-signaling transduction pathways were repressed, whereas the genes associated with cytokinin and jasmonic acid signaling were upregulated for 24-h time point following GA3 treatment. In addition, the floral meristem determinacy genes (JcLFY, JcSOC1) and floral organ identity genes (JcAP3, JcPI, JcSEP1-3) were significantly upregulated, but their negative regulator (JcSVP) was downregulated after GA3 treatment. Moreover, the effects of phytohormone, which was induced by exogenous plant growth regulator, mainly acted on the female floral differentiation process. To the best of our knowledge, this data is the first comprehensive analysis of the underlying transcriptional response mechanism of floral differentiation following GA3 treatment in J. curcas, which helps in engineering high-yielding varieties of Jatropha.

Transcriptome profile analysis reveals the regulation mechanism of floral sex differentiation in Jatropha curcas L.

The seeds of Jatropha curcas contain a high percentage of biodiesel. However, low seed yield which was limited by its poor female flowers was a bottleneck for its utilization. Here, we compared the transcriptomic profiles of five different samples during floral sex differentiation stages using Illumina Hiseq 4000. Our results showed that hundreds of differentially expressed genes (DEGs) were detected in floral sex initiation period, but thousands of DEGs were involved in the stamens and ovules development process. Moreover, the DEGs were mainly shown up-regulation in male floral initiation, but mainly down-regulation in female floral initiation. Male floral initiation was associated with the flavonoid biosynthesis pathway while female floral initiation was related to the phytohormone signal transduction pathway. Cytokinin (CTK) signaling triggered the initiation of female floral primordium, thereafter other phytohormones co-promoted the female floral development. In addition, the floral organ identity genes played important roles in floral sex differentiation process and displayed a general conservation of the ABCDE model in J. curcas. To the best of our knowledge, this data is the first comprehensive analysis of the underlying regulatory mechanism and the related genes during floral sex differentiation in J. curcas, which help in engineering high-yielding varieties of J. curcas.

Identification, Characterization, and Quantification of Impurities of Safinamide Mesilate: Process-Related Impurities and Degradation Products.

The characterization of process-related impurities and degradation products of safinamide mesilate (SAFM) in bulk drug and a stability-indicating HPLC method for the separation and quantification of all the impurities were investigated. Four process-related impurities (Imp-B, Imp-C, Imp-D, and Imp-E) were found in the SAFM bulk drug. Five degradation products (Imp-A, Imp-C, Imp-D, Imp-E, and Imp-F) were observed in SAFM under oxidative conditions. Imp-C, Imp-D, and Imp-E were also degradation products and process-related impurities. Remarkably, one new compound, identified as (S)-2-[4-(3-fluoro-benzyloxy) benzamido] propanamide (i.e., Imp-D), is being reported here as an impurity for the first time. Furthermore, the structures of the aforementioned impurities were characterized and confirmed via IR, NMR, and MS techniques, and the most probable formation mechanisms of all impurities proposed according to the synthesis route. Optimum separation was achieved on an Inertsil ODS-3 column (250 × 4.6 mm, 5 µm), using 0.1% formic acid in water (pH adjusted to 5.0) and acetonitrile as the mobile phase in gradient mode. The proposed method was found to be stability-indicating, precise, linear, accurate, sensitive, and robust for the quantitation of SAFM and its process-related substances, including its degradation products.

Quantitative analysis of ripasudil hydrochloride hydrate and its impurities by reversed-phase high-performance liquid chromatography after precolumn derivatization: Identification of four impurities.

We report the development and validation of a stability-indicating reversed-phase high-performance liquid chromatography method with precolumn derivatization for the separation and identification of the impurities of ripasudil hydrochloride hydrate, a novel protein kinase inhibitor. 2,3,4,6-Tetra-O-acetyl-ß-d-glucopyranosyl isothiocyanate was chosen as the derivatizing reagent and triethylamine was added as catalyst. 200 µL sample solution (1 mg/mL), 600 µL derivatizing reagent (1 mg/mL), and 200 µL triethylamine solution (1%, v/v) were mixed and reacted at 40°C for 30 min. The separation was achieved on an Inertsil C18 ODS-3 (250 mm × 4.6 mm, 5 µm) column using mobile phases including 10 mmol monopotassium phosphate buffer (pH 3.0) and methanol in gradient mode. The column temperature was adjusted at 25°C and the flow rate at 1 mL/min. The detection was carried out at 220 nm. Different precolumn derivatization conditions as well as the high-performance liquid chromatography conditions were optimized. Ripasudil hydrochloride hydrate and its four impurities were detected and quantitated, among which two new compounds were characterized. The proposed method was validated and proven to be selective, accurate, and precise and suitable for the quantitative analysis of ripasudil hydrochloride hydrate.

Identification, characterization and HPLC quantification of process-related impurities in Trelagliptin succinate bulk drug: Six identified as new compounds.

A sensitive, selective and stability indicating reversed-phase LC method was developed for the determination of process related impurities of Trelagliptin succinate in bulk drug. Six impurities were identified by LC-MS. Further, their structures were characterized and confirmed utilizing LC-MS/MS, IR and NMR spectral data. The most probable mechanisms for the formation of these impurities were also discussed. To the best of our knowledge, six structures among these impurities are new compounds and have not been reported previously. The superior separation was achieved on an InertSustain C18 (250mm×4.6mm, 5µm) column in a gradient mixture of acetonitrile and 20mmol potassium dihydrogen phosphate with 0.25% triethylamine (pH adjusted to 3.5 with phosphate acid). The method was validated as per regulatory guidelines to demonstrate system suitability, specificity, sensitivity, linearity, robustness, and stability.