Neuro-stimulating effect of Citri Reticulata Pericarpium Viride essential oil through regulating Glu/NMDAR on olfactory bulb to improve anxiety-like behavior.

ETHNOPHARMACOLOGICAL RELEVANCE: Citri Reticulata Pericarpium Viride (also known Qing-Pi or QP) is a plant in the Rutaceae family, QP is a traditional Qi-regulating medicine in Chinese medicine that is compatible with other Chinese medicine components and has extensive clinical practice in treating anxiety and depression. Reports on the pharmacological effects of QP have demonstrated its neuroprotective effects and antioxidant capacities. Numerous pharmacological benefits of QP are attributed to its antioxidant abilities. Anxiety disorders are a broadly defined category of mental illnesses. Oxidative stress and an imbalance in the antioxidant defense system are typical pathological features of these disorders. AIM OF THE STUDY: The aim of this study was to evaluate the effects of QP essential oil on anxiety using animal models and investigate the underlying neurobiological mechanisms. MATERIALS AND METHODS: This study aimed to develop an animal model of anxiety using chronic restraint stress and investigate the effects of inhalation of Citri Reticulata Pericarpium Viride essential oil on anxiety-like behavior, olfactory function, and olfactory bulb neurogenesis in mice with anxiety. RESULTS: The results showed that long-term chronic restraint stimulation caused a decrease in olfactory function, significant anxiety-like behavior, and a notable reduction in the number of neurons in the olfactory bulb. However, inhalation of Citri Reticulata Pericarpium Viride essential oil reversed these effects, improving the olfactory function, neuro-stimulating effect, alleviating anxiety-like behavior, and regulating theta (4-12Hz) oscillation in the hippocampus DG area. These effects were associated with changes in the expression levels of glutamate receptor NMDAR and NeuN in olfactory bulb. CONCLUSIONS: The study revealed that mice with anxiety induced by chronic restraint stress exhibited significant olfactory dysfunction, providing strong evidence for the causal relationship between anxiety disorders and olfactory dysfunction. Moreover, QP essential oil has the potential to be developed as a therapeutic drug for anxiety disorders, in addition to its role as a complementary anxiolytic.

Homolog of Pea SGR Controls Stay-Green in Faba Bean (Vicia faba L.).

Faba bean is an important legume crop consumed as a vegetable or snack food, and its green cotyledons could present an attractive color for consumers. A mutation in SGR causes stay-green in plants. In this study, vfsgr was identified from a green-cotyledon-mutant faba bean, SNB7, by homologous blast between the SGR of pea and the transcriptome of faba bean. Sequence analysis revealed that a SNP at position 513 of the CDS of VfSGR caused a pre-stop codon, resulting in a shorter protein in the green-cotyledon faba bean SNB7. A dCaps marker was developed according to the SNP that caused the pre-stop, and this marker was completely associated with the color of the cotyledon of faba bean. SNB7 stayed green during dark treatment, while the expression level of VfSGR increased during dark-induced senescence in the yellow-cotyledon faba bean HST. Transient expression of VfSGR in Nicotiana. benthamiana leaves resulted in chlorophyll degradation. These results indicate that vfsgr is the gene responsible for the stay-green of faba bean, and the dCaps marker developed in this study provides a molecular tool for the breeding of green-cotyledon faba beans.

Fine mapping of QTL conferring resistance to calcareous soil in mungbean reveals VrYSL3 as candidate gene for the resistance.

Iron is a crucial nutrient for biological functions in plants. High-pH and calcareous soil is a major stress causing iron deficiency chlorosis (IDC) symptoms and yield losses in crops. Use of calcareous soil-tolerance genetic resources is the most effective preventative method to combat the effects of high-pH and calcareous soils. A previous study using a mungbean recombinant inbred line (RIL) population of the cross Kamphaeg Saen 2 (KPS2; IDC susceptible) × NM-10-12 identified a major quantitative trait locus (QTL), qIDC3.1, which controls resistance and explains more than 40% of IDC variation. In this study, we fine-mapped qIDC3.1 and identified an underlying candidate gene. A genome wide association analysis (GWAS) using 162 mungbean accessions identified single nucleotide polymorphisms (SNPs) on chromosome 6; several SNPs were associated with soil plant analysis development (SPAD) values and IDC visual scores of mungbeans planted on calcareous soil, respectively. These SNPs corresponded to qIDC3.1. Using the same RIL population as in the previous study and an advanced backcross population developed from KPS2 and IDC-resistant inbred line RIL82, qIDC3.1 was further confirmed and fine-mapped to an interval of 217 kilobases harboring five predicted genes, including LOC106764181 (VrYSL3), which encodes a yellow stripe1-like-3 (YSL3) protein, YSL3 is involved in iron deficiency resistance. Gene expression analysis revealed that VrYSL3 was highly expressed in mungbean roots. In calcareous soil, expression of VrYSL3 was significantly up-regulated, and it was more obviously upregulated in the roots of RIL82, than in those of KPS2. Sequence comparison of VrYSL3 between the RIL82 and KPS2 revealed four SNPs that result in amino acid changes in the VrYSL3 protein and a 20-bp insertion/deletion in the promoter where a cis-regulatory element resides. Transgenic Arabidopsis thaliana plants overexpressing VrYSL3 showed enhanced iron and zinc contents in the leaves. Taken together, these results indicate that VrYSL3 is a strong candidate gene responsible for calcareous soil resistance in mungbean.

Identification and Functional Characterization of WRKY, PHD and MYB Three Salt Stress Responsive Gene Families in Mungbean (Vigna radiata L.).

WRKY-, PHD-, and MYB-like proteins are three important types of transcription factors in mungbeans, and play an important role in development and stress resistance. The genes' structures and characteristics were clearly reported and were shown to contain the conservative WRKYGQK heptapeptide sequence, Cys4-His-cys3 zinc binding motif, and HTH (helix) tryptophan cluster W structure, respectively. Knowledge on the response of these genes to salt stress is largely unknown. To address this issue, 83 VrWRKYs, 47 VrPHDs, and 149 VrMYBs were identified by using comparative genomics, transcriptomics, and molecular biology methods in mungbeans. An intraspecific synteny analysis revealed that the three gene families had strong co-linearity and an interspecies synteny analysis showed that mungbean and Arabidopsis were relatively close in genetic relationship. Moreover, 20, 10, and 20 genes showed significantly different expression levels after 15 days of salt treatment (p < 0.05; Log2 FC > 0.5), respectively. Additionally, in the qRT-PCR analysis, VrPHD14 had varying degrees of response to NaCl and PEG treatments after 12 h. VrWRKY49 was upregulated by ABA treatment, especially in the beginning (within 24 h). VrMYB96 was significantly upregulated in the early stages of ABA, NaCl, and PEG stress treatments (during the first 4 h). VrWRKY38 was significantly upregulated by ABA and NaCl treatments, but downregulated by PEG treatment. We also constructed a gene network centered on the seven DEGs under NaCl treatment; the results showed that VrWRKY38 was in the center of the PPI network and most of the homologous Arabidopsis genes of the interacted genes were reported to have response to biological stress. Candidate genes identified in this study provide abundant gene resources for the study of salt tolerance in mungbeans.

Pulsatilla chinensis saponins improve SCFAs regulating GPR43-NLRP3 signaling pathway in the treatment of ulcerative colitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Pulsatilla decoction has been extensively used to treat ulcerative colitis (UC) in recent years. Pulsatilla chinensis saponin (PRS), the active ingredient of its monarch medicine Pulsatilla chinensis (Bunge) Regel, plays a crucial role in the treatment of UC, but its specific mechanism of action has not been fully elucidated. AIM OF THE STUDY: This study aims to investigate the protective effect and possible mechanism of PRS on DSS-induced ulcerative colitis in rats. MATERIALS AND METHODS: In this study, the DSS-induced colitis model was used to explore the metabolism and absorption of PRS under UC, detect the content of short-chain fatty acids (SCFAs) in colon tissue, the expression of receptor G Protein-Coupled Receptor 43 (GPR43) protein and inflammasome NLRP3, and observe the expression level of IL-1ß, IL-6 and TNF-α in colon tissue. The protective effect of the PRS was also observed. RESULTS: It was found that in the UC group, the absorption rate and extent of drugs increased, and the elimination was accelerated. Compared with the control group, PRS increased the content of short-chain fatty acids (SCFAs) in colon tissue, promoted the expression of SCFAs receptor GPR43 protein, inhibited the activation of the NLRP3 inflammasome, and decreased the content of IL-1ß, IL-6 and TNF-α. PRS protects the colon in DSS-induced inflammatory bowel disease by increasing the content of SCFAs, promoting the expression of GPR43 protein, inhibiting the activation of the NLRP3 inflammasome, and reversing the increase in IL-1ß, IL-6 and TNF-α levels. CONCLUSIONS: PRS can increase the content of colonic SCFAs, activate the GPR43-NLRP3 signaling pathway, and reduce the levels of pro-inflammatory cytokines, thereby improving the symptoms of DSS-induced colitis.

Mapping of quantitative trait locus reveals PsXI gene encoding xylanase inhibitor as the candidate gene for bruchid (Callosobruchus spp.) resistance in pea (Pisum sativum L.).

Pea (Pisum sativum L.) is an important legume crop for both food and feed. Bruchids (Callosobruchus spp.) are destructive insect pests of pea in the field and during storage. In this study, we identified a major quantitative trait locus (QTL) controlling seed resistance to C. chinensis (L.) and C. maculatus (Fab.) in field pea using F2 populations derived from a cross between PWY19 (resistant) and PHM22 (susceptible). QTL analysis in the two F2 populations grown in different environments consistently identified a single major QTL, qPsBr2.1, controlling the resistance to both bruchid species. qPsBr2.1 was mapped onto linkage group 2 between DNA markers 18339 and PSSR202109 and explained 50.91% to 70.94% of the variation in resistance, depending on the environment and bruchid species. Fine mapping narrowed down qPsBr2.1 to a genomic region of 1.07 Mb on chromosome 2 (chr2LG1). Seven annotated genes were found in this region, including Psat2g026280 (designated as PsXI), which encodes a xylanase inhibitor and was considered as a candidate gene for bruchid resistance. PCR amplification and sequence analysis of PsXI suggested the presence of an insertion of unknown length in an intron of PWY19, which causes variation in the open reading frame (ORF) of PsXI. Moreover, the subcellular localization of PsXI differed between PWY19 and PHM22. These results together suggested that PsXI encoding xylanase inhibitor is responsible for the bruchid resistance of the field pea PWY19.

Genome-Wide Identification, Expression Analysis, and Potential Roles under Abiotic Stress of the YUCCA Gene Family in Mungbean (Vigna radiata L.).

YUCCA, belonging to the class B flavin-dependent monooxygenases, catalyzes the rate-limiting step for endogenous auxin synthesis and is implicated in plant-growth regulation and stress response. Systematic analysis of the YUCCA gene family and its stress response benefits the dissection of regulation mechanisms and breeding applications. In this study, 12 YUCCA genes were identified from the mungbean (Vigna radiata L.) genome and were named based on their similarity to AtYUCCAs. Phylogenetic analysis revealed that the 12 VrYUCCAs could be divided into 4 subfamilies. The evidence from enzymatic assays in vitro and transgenetic Arabidopsis in vivo indicated that all the isolated VrYUCCAs had biological activity in response to IAA synthesis. Expression pattern analysis showed that functional redundancy and divergence existed in the VrYUCCA gene family. Four VrYUCCAs were expressed in most tissues, and five VrYUCCAs were specifically highly expressed in the floral organs. The response toward five stresses, namely, auxin (indole-3-acetic acid, IAA), salinity, drought, high temperatures, and cold, was also investigated here. Five VrYUCCAs responded to IAA in the root, while only VrYUCCA8a was induced in the leaf. VrYUCCA2a, VrYUCCA6a, VrYUCCA8a, VrYUCCA8b, and VrYUCCA10 seemed to dominate under abiotic stresses, due to their sensitivity to the other four treatments. However, the response modes of the VrYUCCAs varied, indicating that they may regulate different stresses in distinct ways to finely adjust IAA content. The comprehensive analysis of the VrYUCCAs in this study lays a solid foundation for further investigation of VrYUCCA genes' mechanisms and applications in breeding.

Genome-wide association studies provide genetic insights into natural variation of seed-size-related traits in mungbean.

Although mungbean (Vigna radiata (L.) R. Wilczek) is an important legume crop, its seed yield is relatively low. To address this issue, here 196 accessions with 3,607,508 SNP markers were used to identify quantitative trait nucleotides (QTNs), QTN-by-environment interactions (QEIs), and their candidate genes for seed length (SL), seed width, and 100-seed weight (HSW) in two environments. As a result, 98 QTNs and 20 QEIs were identified using 3VmrMLM, while 95, >10,000, and 15 QTNs were identified using EMMAX, GEMMA, and CMLM, respectively. Among 809 genes around these QTNs, 12 were homologous to known seed-development genes in rice and Arabidopsis thaliana, in which 10, 2, 1, and 0 genes were found, respectively, by the above four methods to be associated with the three traits, such as VrEmp24/25 for SL and VrKIX8 for HSW. Eight of the 12 genes were significantly differentially expressed between two large-seed and two small-seed accessions, and VrKIX8, VrPAT14, VrEmp24/25, VrIAR1, VrBEE3, VrSUC4, and Vrflo2 were further verified by RT-qPCR. Among 65 genes around these QEIs, VrFATB, VrGSO1, VrLACS2, and VrPAT14 were homologous to known seed-development genes in A. thaliana, although new experiments are necessary to explore these novel GEI-trait associations. In addition, 54 genes were identified in comparative genomics analysis to be associated with seed development pathway, in which VrKIX8, VrABA2, VrABI5, VrSHB1, and VrIKU2 were also identified in genome-wide association studies. This result provided a reliable approach for identifying seed-size-related genes in mungbean and a solid foundation for further molecular biology research on seed-size-related genes.

Isolation of Phenolic Compounds from Raspberry Based on Molecular Imprinting Techniques and Investigation of Their Anti-Alzheimer's Disease Properties.

Alzheimer's disease is the most common neurodegenerative disease, characterized by memory loss and cognitive dysfunction. Raspberry fruits contain polyphenols which have antioxidant and anti-inflammatory properties. In this study, we used molecular imprinting technology to efficiently isolate phenolic components from the raspberry ethyl acetate extracts. Six phenolic components (ellagic acid, tiliroside, kaempferol-3-o-rutoside, gallic acid, ferulic acid and vanillic acid) were identified by UPLC-Q-TOF-MS analysis. Molecular docking was used to predict the anti-inflammatory effects and anti-Alzheimer's potential of these isolated compounds, which showed a good binding ability to diseases and related proteins. However, the binding energy and docking fraction of ellagic acid, tiliroside, and kaempferol-3-o-rutoside were better than those of gallic acid, ferulic acid and vanillic acid. Additionally, by studying the effects of these six phenolic components on the LPS-induced secretion of inflammatory mediators in murine microglial (BV2) cells, it was further demonstrated that they were all capable of inhibiting the secretion of NO, IL-6, TNF-α, and IL-1ß to a certain extent. However, ellagic acid, tiliroside, and kaempferol-3-o-rutoside have better inhibitory effects compared to others. The results obtained suggest that the phenolic components extracted from ethyl acetate extracts of raspberry by molecularly imprinted polymers have the potential to inhibit the progression of Alzheimer's disease.

Genetic analysis and identification of VrFRO8, a salt tolerance-related gene in mungbean.

Mungbean (Vigna radiata (L.) R. Wilczek) is an important legume crop of Asia. Salt concentrations typically causes major yield reductions in mungbean. Although the biochemical and genetic basis of salt tolerance-related gene are well studied in Arabidopsis and soybean, limited information concerning the salt tolerance-related genes in mungbean. To address this issue, we mined salt tolerance related genes using the survival rate trait and 160,1405 SNPs in 112 mungbean accessions. As a result, VrFRO8 significantly associated with salt-stress were identified in the GWAS analysis. The candidate gene VrFRO8 was evidenced by comparative genomics, transcriptome and RT-qPCR analysis. The expression level of VrFRO8 was significantly up-regulated (P-value = 0.001) after salt treatment compared with the control group. Moreover, 188 genes and 158 transcription factors related to salt-stress signal transduction pathway were mined, and 18 genes (18/188) had higher expression level in the salt-tolerant varieties than salt-sensitive varieties. And, the function of VrFRO8 was predicted in mungbean, the protein interaction between VrFRO8 and seven related-genes were found by molecular structure analysis. VrFRO8 might reduce SOD contents by influence Fe2+/Fe3+ ratio under the damage of salt stress. This study used multi-omics data to mine a key genes significantly associated with salt tolerance, and constructed a VrFRO8-related PPI network for salt tolerance, which would lay a solid foundation for further molecular biology research of VrFRO8 and mungbean breeding.

A Class II KNOX Gene, KNAT7-1, Regulates Physical Seed Dormancy in Mungbean [Vigna radiata (L.) Wilczek].

Seed dormancy in wild mungbean (Vigna radiata var. sublobata) may be useful for the breeding of cultivated mungbean (var. radiata) with pre-harvest sprouting resistance. Previous studies have identified two major quantitative trait loci (QTLs) for seed dormancy, HsA and Sdwa5.1.1+, in wild mungbean that are possibly having the same locus or linked. However, these QTLs have not been confirmed/verified and a molecular basis of seed dormancy in mungbean is not yet known. In this study, we aimed to finely map the Sdwa5.1.1+ and identify candidate gene(s) for this locus. Microscopic observations revealed that wild mungbean "ACC41" seeds had a palisade cuticle layer, while cultivated mungbean "Kamphaeng Saen 2" (KPS2) seeds lacked this layer. Fine mapping using an F2 population developed from a cross between ACC41 and KPS2 revealed two linked QTLs, Sdwa5.1.1+ and Sdwa5.1.2+, controlling seed dormancy. The Sdwa5.1.1+ was confirmed in an F2:3 population derived from the same cross and mapped to a 3.298-Kb region containing only one gene LOC106767068, designated as VrKNAT7-1, which encodes the transcription factor KNOTTED ARABIDOPSIS THALIANA7 (KNAT7), a class II KNOTTED1-LIKE HOMEOBOX (KNOX II) protein. VrKNAX7 sequence alignment between ACC41 and KPS2 revealed several polymorphisms in the coding, untranslated, and promoter regions. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression of VrKNAT7-1 and VrCYP86A, a putative downstream regulation of VrKNAT7-1, in the seed coat of ACC41 is statistically much higher than that of KPS2. Altogether, these results indicate that VrKNAT7-1 controls physical seed dormancy in the wild mungbean ACC41.

Association of 3D-CRT and IMRT accelerated hyperfractionated radiotherapy with local control rate and 5-year survival in esophageal squamous cell carcinoma patients.

OBJECTIVES: This retrospective study examined the relevance and prognostic factors of whole-course conformal radiotherapy (CRT) and late-course accelerated hyperfractionation radiotherapy (LCAFRT) for esophageal squamous cell carcinoma (ESCC). METHODS: A total of 110 patients with ESCC received whole-course CRT and LCAFRT between May 2004 and January 2015. All patients received conventional CRT of 2 Gy per day, up to 30-40 Gy, followed by LCAFRT using reduced fields at 1.5 Gy/fraction twice a day, up to 24-39 Gy, for a total dose of 60-69 Gy. RESULTS: The median follow-up was 85 months. The whole groups 1-, 3-, and 5-year survival rates were 81.8%, 46.4%, and 41.8%, respectively. The local control rates for the whole group at 1, 3, and 5 years were 82.7%, 70.0%, and 68.2%, respectively. There were no significant differences among survival rates and local control rates between the 3D-CRT and intensity-modulated radiotherapy (IMRT) groups. The main reactions to acute radiotherapy were acute radiation tracheitis, esophagitis, and pneumonia. The tumor location and TNM stage were independent prognostic factors for overall survival. CONCLUSION: The results showed that whole-course CRT and LCAFRT for ESCC can improve survival and local control with a tolerable acute reaction compared to previous studies. Local recurrence and distant metastasis are the main failure modes of treatment. ADVANCES IN KNOWLEDGE: Whole-course CRT and LCAFRT for ESCC can improve the survival and local control rate compared with previous studies from the 2DRT era. It might provide another treatment for patients with inoperable ESCC or refusing surgery.

Screening for potential quality markers of Callerya nitida var. hirsutissima. Z.Wei based on components profile, pharmacokinetics, and anti-inflammatory study.

Callerya nitida var. hirsutissima. Z.Wei is a classical, traditional Chinese herbal medicine mostly used to treat rheumatoid arthritis. Recent reports suggest that inconsistent and poor-quality control levels have primarily affected the therapeutic efficacy. Therefore, the aim of the current study was to investigate the active chemical ingredients, stability of components in blood, pharmacokinetics, and pharmacodynamics to specify the potential markers for quality control and quality evaluation of Callerya nitida. The active components in vitro and in vivo were obtained by ultra-high-performance liquid chromatography-mass spectrometry. Moreover, the changes of the bioactive components in the blood were monitored over time (0-24 h) in order to identify stable active components. On this basis, the pharmacokinetic characteristics of these ingredients combined with the anti-inflammatory activity were determined to screen out the potential markers for ensuring the quality control of Callerya nitida. The identified four components, such as calycosin, daidzein, formononetin, and 5-hydroxymethylfurfural, have the characteristics of intrinsic components, clearly defined structures, high exposure values, and in vivo stability, which are important for the therapeutic activity of pharmacologically active materials. Therefore, they can be used as potential markers to control the quality levels of Callerya nitida.

Pulsatilla chinensis Saponins Ameliorate Inflammation and DSS-Induced Ulcerative Colitis in Rats by Regulating the Composition and Diversity of Intestinal Flora.

Pulsatilla chinensis (Bunge) Regel is a commonly used Chinese medicine for clearing away heat and detoxification, cooling blood, stopping dysentery, and anti-inflammatory effects. Pulsatilla chinensis saponins (PRS) have been identified to be responsible for producing these pharmacological activities. Studies have shown that Pulsatilla decoction has a good therapeutic effect on ulcerative colitis (UC), however, the therapeutic effect of PRS on UC has not been reported. Therefore, the purpose of this study was to investigate the possible anti-UC activity of PRS using a dextran sulfate sodium (DSS)-induced rat model, and further study the mechanism of PRS in the treatment of UC. The fecal and colon samples were collected from rats to monitor the changes in the composition and diversity of the intestinal flora, and pathological colon sections were also made to examine the mesenteric hemorheological characteristics. The results showed that PRS significantly reduced the mesenteric blood flow in UC rats and significantly alleviated the inflammatory response, which indicates that saponins are involved in the anti-UC effects of PRS. At the same time, it is also suggested that the regulation of intestinal flora by Pulsatilla chinensis saponins is an important pathway for its anti-UC activity, which may be ascribed to the increase in beneficial bacteria like norank_F_Muribaculaceae and norank_F_norank_O_Clostridia_UCG-014, and decrease in the harmful Bacteroides.

Transcriptomic analysis of salt tolerance-associated genes and diversity analysis using indel markers in yardlong bean (Vigna unguiculata ssp. sesquipedialis).

BACKGROUND: High salinity is a devastating abiotic stresses for crops. To understand the molecular basis of salinity stress in yardlong bean (Vigna unguiculata ssp. sesquipedalis), and to develop robust markers for improving this trait in germplasm, whole transcriptome RNA sequencing (RNA-seq) was conducted to compare the salt-tolerant variety Suzi 41 and salt-sensitive variety Sujiang 1419 under normal and salt stress conditions. RESULTS: Compared with controls, 417 differentially expressed genes (DEGs) were identified under exposure to high salinity, including 42 up- and 11 down-regulated DEGs in salt-tolerant Suzi 41 and 186 up- and 197 down-regulated genes in salt-sensitive Sujiang 1419, validated by qRT-PCR. DEGs were enriched in "Glycolysis/Gluconeogenesis" (ko00010), "Cutin, suberine and wax biosynthesis" (ko00073), and "phenylpropanoid biosynthesis" (ko00940) in Sujiang 1419, although "cysteine/methionine metabolism" (ko00270) was the only pathway significantly enriched in salt-tolerant Suzi 41. Notably, AP2/ERF, LR48, WRKY, and bHLH family transcription factors (TFs) were up-regulated under high salt conditions. Genetic diversity analysis of 84 yardlong bean accessions using 26 InDel markers developed here could distinguish salt-tolerant and salt-sensitive varieties. CONCLUSIONS: These findings show a limited set of DEGs, primarily TFs, respond to salinity stress in V. unguiculata, and that these InDels associated with salt-inducible loci are reliable for diversity analysis.

Single-Molecule Real-Time and Illumina-Based RNA Sequencing Data Identified Vernalization-Responsive Candidate Genes in Faba Bean (Vicia faba L.).

Faba bean (Vicia faba L.) is one of the most widely grown cool season legume crops in the world. Winter faba bean normally has a vernalization requirement, which promotes an earlier flowering and pod setting than unvernalized plants. However, the molecular mechanisms of vernalization in faba bean are largely unknown. Discovering vernalization-related candidate genes is of great importance for faba bean breeding. In this study, the whole transcriptome of faba bean buds was profiled by using next-generation sequencing (NGS) and single-molecule, real-time (SMRT) full-length transcriptome sequencing technology. A total of 29,203 high-quality non-redundant transcripts, 21,098 complete coding sequences (CDS), 1,045 long non-coding RNAs (lncRNAs), and 12,939 simple sequence repeats (SSRs) were identified. Furthermore, 4,044 differentially expressed genes (DEGs) were identified through pairwise comparisons. By Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, these differentially expressed transcripts were found to be enriched in binding and transcription factor activity, electron carrier activity, rhythmic process, and receptor activity. Finally, 50 putative vernalization-related genes that played important roles in the vernalization of faba bean were identified; we also found that the levels of vernalization-responsive transcripts showed significantly higher expression levels in cold-treated buds. The expression of VfSOC1, one of the candidate genes, was sensitive to vernalization. Ectopic expression of VfSOC1 in Arabidopsis brought earlier flowering. In conclusion, the abundant vernalization-related transcripts identified in this study will provide a basis for future researches on the vernalization and faba bean breeding and established a reference full-length transcriptome for future studies on faba bean.

Occurrence of root rot caused by Pythium aphanidermatum on mung bean (Vigna radiata) in China.

In Aug 2019, approximately 10% of mung bean plants at the experimental farm of the Jiangsu Academy of Agricultural Science (32.03 N; 118.88 E) showed symptoms of stunting and wilting. Brown and water-soaked stem lesions were often observed at the base of the diseased plants. In severe cases, the plants collapsed and cumulous aerial mycelia were visible on the basal stem surface (Figure S1 A). To identify the causal agent, a total of 20 tissue fragments (5 mm long) were excised from roots and basal stems of five symptomatic plants. The fragments were surface sterilized in 2% sodium hypochlorite solution then plated on 2.5% potato dextrose agar (PDA) plates containing 10 µg/mL pimaricin, 100 µg/mL ampicillin, 10 µg/mL rifampicin, and 10 µg/mL pentachloronitrobenzene (PARP; Beckerman et al. 2017). After 3-4 days incubation at 25oC in dark, 14 colonies with white and cumulous mycelia grew from the tissue pieces (named as JS19-1 to JS19-14). JS19-1 and JS19-2 were purified by hyphal tipping, then grown on PDA medium for 7 days for morphological observation using a compound microscope (Figure S1 B, C). Width of coenocytic hyphae ranged from 3.7 to 8.9 (avg. 6.1, n=20) µm. Terminal oogonia were globose and with a diameter of 13.8 to 25.8 (avg. 22, n=20) µm. Antheridia were barrel-shaped, and mostly intercalary, sometimes terminal. Most of antheridia were diclinous, with 6.2 to 12.5 (avg. 9.3, n=20) µm in width and 7.6 to 15.3 (avg. 12.8, n=20) µm in length. Oogonia were fertilized with one or two (rare) antheridia. Oospores were aplerotic, 10.1 to 23.5 (avg. 20.4, n=20) µm in diameter. Sporangia had terminal inflated hyphal branches (Figure S1 D, E). The two isolates were preliminary identified as Pythium aphanidermatum. For molecular identification, the sequences of internal transcribed spacer (ITS) rDNA, cytochrome oxidase subunit I (CoxI) (Robideau et al. 2011), and ß-tubulin (Kroon et al. 2004) of JS19-1 were detected, and deposited in GenBank (MT949538, MT949539 and MT949540). The ITS and CoxI sequences were identical with P. aphanidermatum CBS28779 ITS (759/759 bp, HQ643439.1) and PYT01 CoxI (640/640 bp, MH760243.1) respectively, the ß-tubulin sequence showed 99% (830/840 bp) similarity of P. aphanidermatum P2 (AY564048.1). Thus, JS19-1 was confirmed as P. aphanidermatum. To fulfill Koch's postulates, the pathogenicity of JS19-1 was tested using the procedure of Kiyoshi et al. (2021) with some modifications. Barley grains infested with JS19-1 were as inoculum and thoroughly mixed with potting mixture at a rate of 10% in volume. Six mung bean seeds were sown per pot and then grown in a greenhouse. Potting mixture with no inoculum was used as control. Three pots of replicate plants used for inoculation and control. After 3 weeks, emergence in the inoculated pots was 33% and symptoms of stunting and root rot similar to those in field were observed, while control plants were asymptomatic (FigureS1 F, G). P. aphanidermatum was successfully reisolated from symptomatic plants of both methods. The pathogenicity tests were repeated twice. P. aphanidermatum causes seed rot, pre- and postemergence damping-off, or stem/root rot of a wide range of industrial crops and vegetables (Liu et al, 2018). To our knowledge, this is the first report of P. aphanidermatum causing disease on mung bean in China. Since Phytophthora vignae (Sun et al, 2020) and P. myriotylum (Yan et al, 2021) have been reported causing mung bean root rot, integrated disease management should be adopted to reduce damage.

Two polygalacturonase-inhibiting proteins (VrPGIP) of Vigna radiata confer resistance to bruchids (Callosobruchus spp.).

Bruchids (Callosobruchus spp.) are destructive storage pests of mung beans (Vigna radiata). Bruchids infest mature seeds during storage and in the field causing heavy losses. Bruchid resistance in mung bean has been characterized as a dominant trait controlled by a single gene. Several independent mapping studies showed that the Br locus on chromosome 5 was a key quantitative trait loci (QTL) involved in bruchid resistance. Two polygalacturonase-inhibitor protein (PGIP) family genes, VrPGIP1 and VrPGIP2, located in the Br locus may be the primary genes responsible for bruchid resistance in mung bean but no experimental proof is available. We isolated the VrPGIP1 and VrPGIP2 genes from bruchid resistant mung bean cultivar V2802 and purified the proteins by prokaryotic expression. Both VrPGIP1 and VrPGIP2 had polygalacturonase inhibitor activity and both of the PGIP proteins conferred resistance to bruchids in an artificial seed test system. VrPGIPs can inhibit the enzyme activity of polygalacturonase present in males, females and fourth instar larvae of C. maculatus. These results demonstrated that VrPGIP1 and VrPGIP2 play a critical role in bruchid resistance probably through inhibiting polygalacturonase activity.

Fine mapping of QTL conferring Cercospora leaf spot disease resistance in mungbean revealed TAF5 as candidate gene for the resistance.

KEY MESSAGE: This paper reports fine mapping of qCLS for resistance to Cercospora leaf spot disease in mungbean and identified LOC106765332encoding TATA-binding-protein-associated factor 5 (TAF5) as the candidate gene for the resistance Cercospora leaf spot (CLS) caused by the fungus Cercospora canescens is an important disease of mungbean. A QTL mapping using mungbean F2 and BC1F1 populations developed from the "V4718" (resistant) and "Kamphaeng Saen 1" (KPS1; susceptible) has identified a major QTL controlling CLS resistance (qCLS). In this study, we finely mapped the qCLS and identified candidate genes at this locus. A BC8F2 [KPS1 × (KPS1 × V4718)] population developed in this study and the F2 (KPS1 × V4718) population used in a previous study were genotyped with 16 newly developed SSR markers. QTL analysis in the BC8F2 and F2 populations consistently showed that the qCLS was mapped to a genomic region of ~ 13 Kb on chromosome 6, which contains only one annotated gene, LOC106765332 (designated "VrTAF5"), encoding TATA-binding-protein-associated factor 5 (TAF5), a subunit of transcription initiation factor IID and Spt-Ada-Gcn5 acetyltransferase complexes. Sequence comparison of VrTAF5 between KPS1 and V4718 revealed many single nucleotide polymorphisms (SNPs) and inserts/deletions (InDels) in which eight SNPs presented in eight different exons, and an SNP (G4,932C) residing in exon 8 causes amino acid change (S250T) in V4718. An InDel marker was developed to detect a 24-bp InDel polymorphism in VrTAF5 between KPS1 and V4718. Analysis by RT-qPCR showed that expression levels of VrTAF5 in KPS1 and V4718 were not statistically different. These results indicated that mutation in VrTAF5 causing an amino acid change in the VrTAF5 protein is responsible for CLS resistance in V4718.

Mapping and Functional Characterization of Stigma Exposed 1, a DUF1005 Gene Controlling Petal and Stigma Cells in Mungbean (Vigna radiata).

Flowers with exposed stigma increase the outcrossing rate and are useful in developing improved hybrid crop cultivars. This exposure results mainly from the cellular morphology of the petal and pistil, but what affects the formation of the petal and pistil in the late developmental stages is less understood. Here, we characterized a novel floral mutant in mungbean (Vigna radiata), stigma exposed 1 (se1), which displays irregular petals and pistils. Floral organ initiation in the se1 mutant was normal, but petal and pistil growth malfunctioned during late development. A histological analysis revealed that the se1 mutant had wrinkled petals with knotted structures and elongated styles. The cellular morphology of the epidermal layers of the se1 petals was deformed, while the cell lengths in the styles increased. A genetic analysis indicated that the se1 phenotype is controlled by a single recessive gene, and it was mapped to chromosome 11. A sequence analysis suggested that a DUF1005-encoding gene, LOC106777793, is the gene controlling the se1 phenotype. The se1 mutant possessed a single-nucleotide polymorphism that resulted in an amino acid change in VrDUF1005. Overexpression of VrDUF1005 in Arabidopsis resulted in rolling leaves and reduced floral size. Consequently, we proposed that VrSE1 functions to modulate cell division in petals and cell expansion in styles during the late developmental stages in mungbean. The se1 mutant is a new genetic resource for mung bean hybrid breeding.