Genome-wide analysis of the mulberry (Morus abla L.) GH9 gene family and the functional characterization of MaGH9B6 during the development of the abscission zone.

Plant glycoside hydrolase family 9 genes (GH9s) are widely distributed in plants and involved in a variety of cellular and physiological processes. In the current study, nine GH9 genes were identified in the mulberry and were divided into two subfamilies based on the phylogenetic analysis. Conserved motifs and gene structure analysis suggested that the evolution of the two subfamilies is relatively conserved and the glycoside hydrolase domain almost occupy the entire coding region of the GH9s gene. Only segmental duplication has played a role in the expansion of gene family. Collinearity analysis showed that mulberry GH9s had the closest relationship with poplar GH9s. MaGH9B1, MaGH9B6, MaGH9B5, and MaGH9B3 were detected to have transcript accumulation in the stalk of easy-to drop mature fruit drop, suggesting that these could play a role in mulberry fruit drop. Multiple cis-acting elements related to plant hormones and abiotic stress responses were found in the mulberry GH9 promoter regions and showed different activities under exogenous abscisic acid (ABA) and 2,4- dichlorophenoxyacetic acid (2,4-D) stresses. We found that the lignin content in the fruit stalk decreased with the formation of the abscission zone (AZ), which could indirectly reflect the formation process of the AZ. These results provide a theoretical basis for further research on the role of GH9s in mulberry abscission.

MaABI5 and MaABF1 transcription factors regulate the expression of MaJOINTLESS during fruit abscission in mulberry (Morus alba L.).

Mulberry holds significant economic value. However, during the ripening stage of its fruit, the phenomenon of abscission, resulting in heavy fruit drop, can severely impact the yield. The formation of off-zone structures is a critical factor in the fruit abscission process, and this process is regulated by multiple transcription factors. One such key gene that plays a significant role in the development of the off-zone in the model plant tomato is JOINTLESS, which promotes the expression of abscission-related genes and regulates the differentiation of abscission zone tissue cells. However, there is a lack of information about fruit abscission mechanism in mulberry. Here, we analyzed the MaJOINTLESS promoter and identified the upstream regulators MaABF1 and MaABI5. These two regulators showed binding with MaJOINTLESS promoter MaABF1 (the ABA Binding Factor/ABA-Responsive Element Binding Proteins) activated the expression of MaJOINTLESS, while MaABI5 (ABSCISIC ACID-INSENSITIVE 5) inhibited the expression of MaJOINTLESS. Finally, the differentially expressed genes (DEGs) were analyzed by transcriptome sequencing to investigate the expression and synergistic relationship of endogenous genes in mulberry during abscission. GO classification and KEGG pathway enrichment analysis showed that most of the DEGs were concentrated in MAPK signaling pathway, flavonoid biosynthesis, citric acid cycle, phytohormone signaling, amino acid biosynthesis, and glycolysis. These results provide a theoretical basis for subsequent in-depth study of physiological fruit abscission in mulberry.

Integrating transcriptome and metabolome analyses of the response to cold stress in pumpkin (Cucurbita maxima).

Cucurbita maxima belong to the genus Cucurbita and are of nutritional and economic importance. Physiological activity, transcriptome, and metabolome analyses of leaf samples from the C. maxima inbreding line IL7 treated at 5 °C and 25 °C were performed. Cold stress resulted in a significant increase in the malondialdehyde content, relative electrical conductivity, soluble protein, sugar content, and catalase activity. A total of 5,553 differentially expressed genes were identified, of which 2,871 were up-regulated and 2,682 down-regulated. In addition, the transcription of differentially expressed genes in the plant hormone signal transduction pathway and transcription factor families of AP2/ERF, bHLH, WRKY, MYB, and HSF was activated. Moreover, 114 differentially expressed metabolites were identified by gas chromatography time-of-flight mass spectrometry, particularly through the analysis of carboxylic acids and derivatives, and organooxygen compounds. The demonstration of a series of potential metabolites and corresponding genes highlighted a comprehensive regulatory mechanism. These findings will provide novel insights into the molecular mechanisms associated with the response to cold stress in C. maxima.

[Conversion method for linear retention indices in gas chromatography acquired using n-alkanes and n-fatty acid methyl esters].

Retention index (RI) is a powerful tool for gas chromatography-based structure elucidation of various compounds. In this study, linear RIs based on n-alkanes and n-fatty acid methyl esters in different temperature programs were fitted. Nearly identical first-order linearities were acquired with different temperature programs. The linear equations were y=1.0051x+318.51 (r2=1) and y=1.0362x+562.519 (r2=1) for polar (packed with polyethylene glycol) and semi-standard non-polar (packed with 5% diphenyl/95% dimethylpolysiloxane) columns, respectively. The variables x and y in the equations stand for RIs based on n-fatty acid methyl esters and n-alkanes, respectively. The established linearity was then verified using the reference RIs collected in the NIST mass spectral library, Golm metabolite library, and Fiehnmetabolite library. The RI values converted using both the acquired equations finely matched those in the references. The established relationship between the two kinds of RIs can be used to expand the RI references and decrease the number of RI acquisition experiments.

[Determination of maleic hydrazide and its glucosides in tobacco leaves using hydrophilic interaction liquid chromatography-tandem mass spectrometry].

A hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method was established for the simultaneous determination of maleic hydrazide (MH) and its two glucosides in tobacco leaves. Ultrasonic assisted extraction of MH and its glucosides was performed using acetonitrile-methyl tert-butyl ether-water (7:10:13, volume ratio). The extraction solution was then centrifuged, and the subnatant was transferred for solvent replacement using acetonitrile. The extract in acetonitrile was then analyzed using HILIC-MS/MS. Method validation was performed, and the linear ranges for MH and MH-O-ß -D-glucoside were 5-150 mg/kg with correlation coefficients (r2) greater than 0.9971 for matrix-matched calibration curves. Limits of detection for MH and MH-O-ß -D-glucoside were 0.5 mg/kg and 0.3 mg/kg, and limits of quantification were 1.0 mg/kg and 0.8 mg/kg, respectively. The recoveries were in the range of 83.1%-112.3% at three spiked levels (10, 40, 80 mg/kg), with intra-day repeatability of 2.7% and 3.8%, inter-day repeatability of 8.3% and 7.1% at 40 mg/kg. The established method was used for the study of MH metabolism in tobacco leaves. By the 28th day after MH spraying, the content of MH in tobacco leaves had decreased by 80.8%, of which only 7.6% transformed to MH-glucosides. The disposition of the remainder needs to be studied.

[Spatial and temporal characteristics of flue-cured tobacco water requirement and irrigation requirement index in Yunnan Province, China].

Based on the daily meteorological data of 124 agricultural meteorological sites during 1977-2010 in Yunnan Province, using recommended Penman-Monteith formula by FAO, water requirement and irrigation requirement index in the growth period of flue-cured tobacco were calculated to analyze their spatial and temporal characteristics and change patterns. The results showed that water requirements of flue-cured tobacco in root extending, vigorous, mature periods and field growth period during 1977-2010 were 76.73-174.73, 247.50-386.64, 180.28-258.14 and 528.18-764.08 mm, respectively, and the water requirement of vigorous period was the highest. The average irrigation demand index of each period was -0.02, 0.38, 0.17 and 0.26, respectively. Effective precipitation could meet the demand of flue-cured tobacco in root extending period. Water requirement of flue-cured tobacco in Yunnan Province decreased annually, and the rates of water requirement under the climate change trend in the four periods abovementioned were -12. 42, -21.46, -7.17 and -47.15 mm . (10 a)-1, respectively. The smallest irrigation demand index was observed in Dehong, and the largest in Diqing. The irrigation demand indexes of Dehong, Xishuangbanna and Puer regions were negative in flue-cured tobacco field growth period. The reference crop evapotranspiration, water requirement and effective precipitation decreased, but the irrigation requirement and irrigation requirement index increased with the increase of latitude. The effective precipitation decreased, but the irrigation requirement and irrigation requirement index increased with the increase of altitude.

Simultaneous determination of alkaloids and their related tobacco-specific nitrosamines in tobacco leaves using LC-MS-MS.

Tobacco alkaloids (e.g., nicotine) and their metabolized tobacco-specific nitrosamines (TSNAs) are very important compounds for tobacco quality and safety. A simple and specific liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of eight tobacco alkaloids and their related four TSNAs in tobacco leaves. The milled tobacco was extracted using 0.1 mol/L ammonium acetate solution and purified using methanol. Mass spectrometry parameters including declustering potential and collision energy were optimized to ensure that both the TSNAs and the tobacco alkaloids have suitable responses. Recoveries for accuracy were in the range of 80.2-105.2%. Intra-day and inter-day repeatability were in the range of 1.7-12.1% and 6.4-18.7%, respectively. Limit of detection and limit of quantitation were estimated in the range of 6 ng/g-45 µg/g and 24 ng/g-90 µg/g, respectively. The established method was applied to investigate the distribution of tobacco alkaloids and TSNAs in four kinds of tobacco. The result showed that the burley and the flue-cured have the highest (0.00047%) and the lowest (0.000024%) percentage of transformation from alkaloids to TSNAs, respectively. Thus, this method can be used for a wide range of samples.

EUS-guided ethanol ablation of insulinomas: case series and literature review.

Surgical resection is a standard treatment for insulinomas; however, it is associated with a high risk of complications and limited to specific suitable candidates. In recent years, endoscopic ultrasound (EUS)-guided ethanol ablation of insulinomas has emerged as a new therapeutic option, especially for elderly patients and candidates unfit for surgery. We aimed to evaluate the feasibility and safety of this technique for insulinomas. Four patients diagnosed with insulinomas based on EUS-fine-needle aspiration and immunohistochemistry results underwent EUS-guided 95% ethanol ablation. A comprehensive literature review was performed to understand the current status of the feasibility, safety, and effects of EUS-guided ethanol ablation of insulinomas. EUS-guided ethanol ablation of insulinomas was successfully completed in all the 4 patients. There were no perioperative or postoperative complications. The patients were discharged at 3 days after the procedure. No recurrence of hypoglycemia or tumors was noted during follow-up (range, 3-6 months). Literature review showed 8 patients with insulinomas who underwent EUS-guided ethanol ablation. All the procedures were successful, with no need for further surgical treatment. Among these reviewed cases, 6 patients had no post-procedural complications, while other 2 patients showed a mild increase in the serum levels of lipase and/or pancreatic enzymes within 48 h post-procedure; furthermore, 1 of these 2 patients presented at a later date with medically controllable hematoma and ulceration. During follow-up, 6 patients remained asymptomatic and normoglycemic, while the 2 patients who presented post-procedural complications developed occasional mild confusion. EUS-guided ethanol ablation of insulinomas is an effective and safe modality, with an acceptable level of post-procedural complications. However, the long-term effects of this new therapeutic option need to be validated in a large randomized controlled trial with longer follow-up.

Liquid chromatography with mass spectrometry method based two-step precursor ion scanning for the structural elucidation of flavonoids.

Plant flavonoids are very important secondary metabolites for insect and virus control of their host plant and are potent nutrients for humans. To be able to understand the bioavailability and functions of plant flavonoids, it is necessary to reveal their exact chemical structures. Liquid chromatography with tandem mass spectrometry is a powerful approach for structural elucidation of metabolites. In this report, a two-step precursor ion scanning based liquid chromatography with tandem mass spectrometry method was developed for the structural elucidation of plant flavonoids. The established method consists of the two-step precursor ions scanning for possible flavonoids extraction, MS(2) fragment spectra acquisition and comparison with an online database, liquid chromatography retention rules correction, and commercial standards verification. The developed method was used for the structure elucidation of flavonoids in flowers and leaves of tobacco (Nicotiana tabacum), and 17 flavonoids were identified in the tobacco variety Yunyan 97. Nine of the 17 identified flavonoids were considered to be found in tobacco flowers or/and leaves for the first time based on the available references. This method was proved to be very effective and can be used for the identification of flavonoids in other plants.

A diverse set of miRNAs responsive to begomovirus-associated betasatellite in Nicotiana benthamiana.

BACKGROUND: Roles of microRNAs (miRNAs) and short interfering RNAs (siRNAs) in biotic stress responses, e.g., viral infection, have been demonstrated in plants by many studies. Tomato yellow leaf curl China virus (TYLCCNV) is a monopartite begomovirus that can systemically infect Solanaceae plants, and induces leaf curling, yellowing and enation symptoms when co-inoculated with a betasatellite (TYLCCNB). The released genome sequence of Nicotiana benthamiana provides an opportunity to identify miRNAs and siRNAs responsive to begomovirus-associated betasatellite in N. benthamiana. RESULTS: miRNAs were identified in three small RNA libraries generated using RNA isolated from N. benthamiana plants systemically infected with TYLCCNV (Y10A) alone, co-infected with Y10A and its betasatellite TYLCCNB (Y10ß) or a TYLCCNB mutant (Y10mß) that contains a mutated ßC1, the sole betasatellite-encoded protein. A total of 196 conserved miRNAs from 38 families and 197 novel miRNAs from 160 families were identified. Northern blot analysis confirmed that expression of species-specific miRNAs was much lower than that of conserved miRNAs. Several conserved and novel miRNAs were found to be responsive to co-infection of Y10A and Y10ß but not to co-infection of Y10A and Y10mß, suggesting that these miRNAs might play a role unique to interaction between Y10ß and N. benthamiana. Additionally, we identified miRNAs that can trigger the production of phased secondary siRNAs (phasiRNAs). CONCLUSIONS: Identification of miRNAs with differential expression profiles in N. benthamiana co-infected with Y10A and Y10ß and co-infected with Y10A and Y10mß indicates that these miRNAs are betasatellite-responsive. Our result also suggested a potential role of miRNA-mediated production of phasiRNAs in interaction between begomovirus and N. benthamiana.

Establishment of a novel method for primary culture of normal human cervical keratinocytes.

BACKGROUND: Cervical keratinocytes are recovered at a low numbers and frequently associated with contaminating human fibroblasts which rapidly overgrow the epithelial cells in culture with medium supplemented with 10% fetal bovine serum (FBS). However, it is difficult to initiate keratinocyte cultures with serum-free keratinocyte growth medium alone because cell attachment can be poor. Therefore, the culture of these cells is extremely difficult. In this study, we described a modified culture medium and coated culture plastics for growing normal human cervical epithelial cells in vitro. METHODS: Normal cervical epithelial tissue pieces were obtained and digested with type I collagenase to dissociate the cells and a single cell suspension produced. The cells were cultured on plastic tissue culture substrate alone or substrate coated with collagen type I from rat tail, with modified keratinocyte serum-free medium (K-SFM) supplemented with 5% FBS. After attachment, the medium were replaced with K-SFM without FBS. The expression of basal keratins of the ectocervical epithelium, K5, K14 and K19 were assayed by immunofluorescence with monoclonal antibodies to identify the cell purity. RESULTS: Our results indicate that cells attached to the culture plastic more quickly in K-SFM supplemented with 5% FBS than in K-SFM alone, as well as to tissue culture plastic coated with collagen type I than plastic alone. The modified medium composed of K-SFM and 5% FBS combined with a specific tissue culture plastic coated with collagen type I from rat tail was the best method for culture of normal cervical epithelial cells. K5, K14 and K19 were assayed and keratinocyte purity was nearly 100%. CONCLUSION: A novel, simple and effective method can be used to rapidly obtain highly purified keratinocytes from normal human cervical epithelium.

Three novel xanthones from Garcinia paucinervis and their anti-TMV activity.

Phytochemical investigations of the leaves of Garcinia paucinervis resulted in the isolation of three new xanthones 1-3 and five known analogues 4-8. Structural elucidations of 1-3 were performed by spectral methods such as 1D and 2D (HMQC, HMBC, and ROESY) NMR spectroscopy, in addition to high resolution mass spectrometry. Compounds 1-3 showed anti-TMV activities, with inhibition rates above 20%, especially for 1, which had a lower IC50 value of 21.4 µM.

Diversity arrays technology (DArT) for studying the genetic polymorphism of flue-cured tobacco (Nicotiana tabacum).

Diversity arrays technology (DArT) is a microarray-based marker system that achieves high throughput by reducing the complexity of the genome. A DArT chip has recently been developed for tobacco. In this study, we genotyped 267 flue-cured cultivars/landraces, including 121 Chinese accessions over five decades from widespread geographic regions in China, 103 from the Americas, and 43 other foreign cultivars, using the newly developed chip. Three hundred and thirty polymorphic DArT makers were selected and used for a phylogenetic analysis, which suggested that the 267 accessions could be classified into two subgroups, which could each be further divided into 2‒4 sections. Eight elite cultivars, which account for 83% of the area of Chinese tobacco production, were all found in one subgroup. Two high-quality cultivars, HHDJY and Cuibi1, were grouped together in one section, while six other high-yield cultivars were grouped into another section. The 330 DArT marker clones were sequenced and close to 95% of them are within non-repetitive regions. Finally, the implications of this study for Chinese flue-cured tobacco breeding and production programs were discussed.

Genome sequence of the tobacco bacterial wilt pathogen Ralstonia solanacearum.

Ralstonia solanacearum is a causal agent of plant bacterial wilt with thousands of distinct strains in a heterogeneous species complex. Here we report the genome sequence of a phylotype IB strain, Y45, isolated from tobacco (Nicotiana tabacum) in China. Compared with the published genomes of eight strains which were isolated from other hosts and habitats, 794 specific genes and many rearrangements/inversion events were identified in the tobacco strain, demonstrating that this strain represents an important node within the R. solanacearum complex.

Cloning, structural features, and expression analysis of resistance gene analogs in tobacco.

Using degenerate primers based on the conserved nucleotide binding site (NBS) and protein kinase domain (PKD), 100 resistance gene analogs (RGAs) were isolated from tobacco variety Nicotiana repanda. BLASTx search against the GenBank database revealed that 27 belong to the NBS class and 73 belong to the protein kinase (PK) class. Cluster analysis and multiple sequence alignment of the deduced protein sequences indicate that RGAs of the NBS class can be divided into two groups: toll/interleukin receptor (TIR) and non-TIR types. Both types possess 6 conserved motifs (P-loop, RNBS-A, Kinase-2, RNBS-B, RNBS-C, GLPL). Based on their sequence similarity, the tobacco RGAs of the PK class were assigned to 8 subclasses. We examined their expression after infection with either Tobacco mosaic virus (TMV) or the tobacco black shank pathogen (Phytophthora parasitica var. nicotianae). The expression levels of 4 RGAs of the PK class were significantly elevated by TMV and 1 RGA of the PK class and 3 RGAs of the NBS class were up-regulated by P. parasitica var. nicotianae. The expression of two RGAs of the PK class was induced by P. parasitica var. nicotianae. Infection by either TMV or P. parasitica var. nicotianae enhanced the expression of NtRGA2, a RGA of the PK class. The present study shows that RGAs are abundant in the tobacco genome and the identification of tobacco RGAs induced by pathogens should provide valuable information for cloning related resistance genes in tobacco.

[Genetic and correlation analysis for agronomic traits in flue-cured tobacco (Nicotiana tabacum L.)].

By using a genetic model including additive and dominance effects and their interaction with environments, 7 agronomic traits were analyzed for a diallel design in 4 environmental conditions with 14 flue-cured tobacco varieties (or breeding lines) and their 41 F1 crosses. It was revealed that additive effects were the major genetic component for plant height, internode length, and width of leaves. Number of leaves and length of leaves were mainly controlled by dominance x environment interaction effects. Additive x environment interaction effects and dominance x environment interaction effects played a major role for girth of stem. Yield performance was mainly controlled by additive effects and dominance x environment interaction effects. The varieties adapted to local environments tended to have highly positive additive effects. Dominance effects and the dominance x environment interaction effects could perform differently in positive or negative direction for many crosses. The breeding program for hybrids should consider the adaptation of hybrids to specific ecological environments. The analysis of correlation among agronomic traits indicated that most of phenotypic, genotypic, additive and dominance correlation coefficients were positive. Additive correlations were predominance in genetic correlations for most pairs of traits. Yield can be improved by indirect selection on plant height.

[Genetic contribution of agronomic traits to yield in flue-cured tobacco (Nicotiana tabacum L.)].

In order to understand the genetic contribution of six agronomic traits to yield, 14 flue-cured tobacco varieties (or breeding lines) and their 41 F1 crosses were used for multivariable conditional analysis. The contribution of additive variance of plant height to yield was larger than other agronomic traits. The largest contribution of dominant variance to yield was due to the length of middle leaves. All agronomic traits investigated had small contribution to yield due to additive x environment interaction effects and dominant x environment interaction effects. No identical trait of different parents showed the largest contribution to additive effect of yield. This could be resulted from the fact that each parent had its own genetic and developmental characterization. The dominant effects of yield were mainly influenced by length of middle leaves in most crosses. Length of middle leaves could be served as ameasurement to indirectly select the cross parent having high dominant effect of yield.