Salvia miltiorrhiza ameliorates endometritis in dairy cows by relieving inflammation, energy deficiency and blood stasis.

Introduction: According to traditional Chinese veterinary medicine, endometritis is caused by a combination of Qi deficiency, blood stasis, and external evil invasion. Salvia miltiorrhiza is a traditional Chinese medicine that counteracts blood stasis and has additional demonstrated effects in boosting energy and restraining inflammation. Salvia miltiorrhiza has been employed in many traditional Chinese prescriptions that have proven effective in healing clinical dairy cow endometritis. Methods: the in vivo effect of Salvia miltiorrhiza in treating endometritis was evaluated in dairy cows. In addition, bovine endometrial epithelium cell inflammation and rat blood stasis models were employed to demonstrate the crosstalk between energy, blood circulation and inflammation. Network analysis, western blotting, qRT-PCR and ELISA were performed to investigate the molecular mechanism of Salvia miltiorrhiza in endometritis treatment. Results: The results demonstrate that treatment with Salvia miltiorrhiza relieves uterine inflammation, increases blood ATP concentrations, and prolongs blood clotting times. Four of the six Salvia miltiorrhiza main components (SMMCs) (tanshinone IIA, cryptotanshinone, salvianolic acid A and salvianolic acid B) were effective in reversing decreased ATP and increased IL-1ß, IL-6, and IL-8 levels in an in vitro endometritis model, indicating their abilities to ameliorate the negative energy balance and external evil invasion effects of endometritis. Furthermore, in a blood stasis rat model, inflammatory responses were induced in the absence of external infection; and all six SMMCs inhibited thrombin-induced platelet aggregation. Network analysis of SMMC targets predicted that Salvia miltiorrhiza may mediate anti-inflammation via the Toll-like receptor signaling pathway; anti-aggregation via the Platelet activation pathway; and energy balance via the Thermogenesis and AMPK signaling pathways. Multiple molecular targets within these pathways were verified to be inhibited by SMMCs, including P38/ERK-AP1, a key molecular signal that may mediate the crosstalk between inflammation, energy deficiency and blood stasis. Conclusion: These results provide mechanistic understanding of the therapeutic effect of Salvia miltiorrhiza for endometritis achieved through Qi deficiency, blood stasis, and external evil invasion.

Identification of CmbHLH Transcription Factor Family and Excavation of CmbHLHs Resistant to Necrotrophic Fungus Alternaria in Chrysanthemum.

Chrysanthemum morifolium Ramat. 'Huaihuang' is a traditional Chinese medicinal plant. However, a black spot disease caused by Alternaria sp., a typical necrotrophic fungus, has a serious damaging influence on the field growth, yield, and quality of the plant. 'Huaiju 2#' being bred from 'Huaihuang', shows resistance to Alternaria sp. bHLH transcription factor has been widely studied because of their functions in growth development, signal transduction, and abiotic stress. However, the function of bHLH in biotic stress has rarely been studied. To characterize the resistance genes, the CmbHLH family was surveyed in 'Huaiju 2#'. On the basis of the transcriptome database of 'Huaiju 2#' after Alternaria sp. inoculation, with the aid of the Chrysanthemum genome database, 71 CmbHLH genes were identified and divided into 17 subfamilies. Most (64.8%) of the CmbHLH proteins were rich in negatively charged amino acids. CmbHLH proteins are generally hydrophilic proteins with a high aliphatic amino acid content. Among the 71 CmbHLH proteins, five CmbHLHs were significantly upregulated by Alternaria sp. infection, and the expression of CmbHLH18 was the most significant. Furthermore, heterologous overexpression of CmbHLH18 could improve the resistance of Arabidopsis thaliana to necrotrophic fungus Alternaria brassicicola by enhancing callose deposition, preventing spores from entering leaves, reducing ROS accumulation, increasing the activities of antioxidant enzymes and defense enzymes, and promoting their gene expression levels. These results indicate that the five CmbHLHs, especially CmbHLH18, may be considered candidate genes for resistance to necrotrophic fungus. These findings not only increase our understanding of the role CmbHLHs play in biotic stress but also provide a basis by using CmbHLHs to breed a new variety of Chrysanthemum with high resistance to necrotrophic fungus.

Integrated volatile metabolomic and transcriptomic analysis provides insights into the regulation of floral scents between two contrasting varieties of Lonicera japonica.

Lonicera japonica Thunb., belonging to the Caprifoliaceae family, is an important traditional Chinese medicinal plant. The L. japonica flower (LJF) is widely used in medicine, cosmetics, drinks, and food due to its medicinal and sweet-smelling properties. Considerable efforts have been devoted to investigating the pharmacological activities of LJF; however, the regulatory mechanism of the floral scents remains unknown. We previously selected and bred an elite variety of L. japonica var. chinensis Thunb. called 'Yujin2', which has a strong aroma and is used in functional drinks and cosmetics. In order to reveal the regulatory mechanism of the floral scents of LJF, volatile metabolomic and transcriptomic analyses of the LJF at the silver flowering stage of 'Yujin2' (strong aroma) and 'Fengjin1' (bland odor) were performed. Our results revealed that a total of 153 metabolites and 9,523 genes were differentially regulated in LJF between 'Yujin2' and 'Fengjin1'. The integrated analysis of omics data indicated that the biosynthetic pathways of terpenoids (i.e., monoterpenoids, including geraniol and alpha-terpineol; sesquiterpenoids, including farnesol, farnesal, and alpha-farnesene; triterpenoid squalene), tryptophan and its derivatives (methyl anthranilate), and fatty acid derivatives, were major contributors to the stronger aroma of 'Yujin2' compared to 'Fengjin1'. Moreover, several genes involved in the terpenoid biosynthetic pathway were characterized using quantitative real-time PCR. These results provide insights into the metabolic mechanisms and molecular basis of floral scents in LJF, enabling future screening of genes related to the floral scent regulation, such as alpha-terpineol synthase, geranylgeranyl diphosphate synthase, farnesyl pyrophosphate synthase, anthranilate synthase, as well as transcription factors such as MYB, WRKY, and LFY. The knowledge from this study will facilitate the breeding of quality-improved and more fragrant variety of L. japonica for ornamental purpose and functional beverages and cosmetics.

The integration of transcriptomic and transgenic analyses reveals the involvement of the SA response pathway in the defense of chrysanthemum against the necrotrophic fungus Alternaria sp.

Chrysanthemum morifolium cv. 'Huaihuang' has ornamental, edible, medicinal, and tea product uses. However, its field growth, yield, and quality are negatively affected by black spot disease caused by Alternaria sp. (Strain: HQJH10092301; GenBank accession number: KF688111). In this study, we transcriptionally and transgenically characterized a new cultivar, 'Huaiju 2#' (Henan Traditional Chinese Medicine Plant Cultivar identification number: 2016002), which was bred from 'Huaihuang' and shows resistance to Alternaria sp. Numerous 'Huaiju 2#' plants were inoculated with Alternaria sp. for three or five days. Metabolic analysis showed increases in both salicylic acid (SA) and jasmonic acid (JA) in infected plants compared to the control. Protein activity analysis also revealed a significant increase in defense enzyme activities in infected plants. RNA-Seq of plants infected for 3 or 5 days produced a total of 58.6 GB of clean reads. Among these reads, 16,550 and 13,559 differentially expressed genes (DEGs) were identified in Cm_3 dpi (sample from 3 days post-inoculation labeled as Cm_3 dpi) and Cm_5 dpi (sample from 5 days post-inoculation labeled as Cm_5 dpi), respectively, compared with their controls (Cm_0 d: a mixture samples from 0 d (before inoculation) and those treated with sterile distilled water at 3 dpi and 5 dpi). Gene annotation and cluster analysis of the DEGs revealed a variety of defense responses to Alternaria sp. infection, which were characterized by increases in resistance (R) proteins and the reactive oxygen species (ROS), Ca2+, mitogen-activated protein kinase (MAPK), and JA signaling pathways. In particular, SA signaling was highly responsive to Alternaria sp. infection. The qPCR analysis of 12 DEG candidates supported their differential expression characterized by using the RNA-Seq data. One candidate was CmNPR1 (nonexpressor of pathogenesis-related gene 1), an important positive regulator of SA in systemic acquired resistance (SAR). Overexpression of CmNPR1 in 'Huaiju 2#' increased the resistance of transgenic plants to black spot. These findings indicate that the SA response pathway is likely involved in the defense of 'Huaiju 2#' against Alternaria sp. pathogens.

Identification and Validation of Reference Genes for qRT-PCR Studies of Gene Expression in Dioscorea opposita.

Quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most common methods for gene expression studies. Data normalization based on reference genes is essential for obtaining reliable results for qRT-PCR assays. This study evaluated potential reference genes of Chinese yam (Dioscorea opposita Thunb.), which is an important tuber crop and medicinal plant in East Asia. The expression of ten candidate reference genes across 20 samples from different organs and development stages was assessed. We identified the most stable genes for qRT-PCR studies using combined samples from different organs. Our results also suggest that different suitable reference genes or combinations of reference genes for normalization should be applied according to different organs and developmental stages. To validate the suitability of the reference genes, we evaluated the relative expression of PE2.1 and PE53, which are two genes that may be associated with microtuber formation. Our results provide the foundation for reference gene(s) selection in D. opposita and will contribute toward more accurate gene analysis studies of the genus Dioscorea.

Cryopreservation of plantlet nodes of Dioscorea opposita Thunb. using a vitrification method.

A cryopreservation method by vitrification was developed for long-term storage of Dioscorea opposita Thunb., a valuable native medicinal plant species in Henan Province of China. The cryopreservation protocol was established with cultivar B and evaluated with another four cultivars, Tiegun, 47, Taigu and Huaiqing 1. The results showed that nodes with a bud excised from 60 d plantlets were desirable for the cryopreservation. The optimum procedure was established as: 1) the plantlets were cultured on the Murashige and Skoog (MS) medium supplemented with 2 mg L(-1) KT and 0.02 mg L(-1) NAA at 4 degree C for 7 d before nodes with length of 1-1.5 cm were excised; 2) the nodes were precultured at 4 degree C for 7 d on the MS supplemented with 10 percent dimethyl sulfoxide (DMSO) followed by loading with 60 percent Dioscorea vitrification solution 1 (DVS1): 22 percent (w/v) glycerol + 13 percent (w/v) ethylene glycol + 13 percent (w/v) polyethylene glycol + 10 percent (w/v) DMSO for 60 min at 0 degree C and dehydrated with 100 percent DVS1 for 60 min at 0 degree C; 3) the nodes were then immersed into liquid nitrogen (LN) directly and conserved for 180 d; 4) after rapid thawing in a water-bath at 37 degree C, the nodes were rinsed four times with MS medium supplemented with 5 percent sucrose, then transferred to the MS medium supplemented with 2 mg L(-1) kinetin (KT) and 0.02 mg L(-1) NAA for regeneration. In the present research the regeneration rate of cv. B was about 77.1 percent, those of cvs. Tiegun and Huaiqing 1 were 67.2 percent and 54.0 percent respectively, while cvs. Taigu and 47 were about 40 percent. There were no visual changes observed between the plantlets regenerated from nodes with and without cryopreservation in terms of the morphology indices, indicating that the method established could be applicable to D. opposita with optimized protocol.

[Effect of medium components on callus induction and contents of polysaccharides in calli from Achyranthes bidentata].

OBJECTIVE: To study the effect of medium components on the callus induction and the contents of polysaccharides in calli from Achyranthes bidentata. METHOD: Leaves and stems were selected as explants. The effects of six kinds of factors including basal culture medium, carbon source, 2,4-D, 6-BA, TDZ, CH on the callus induction and the contents of ABPS in calli on the high growth point were studied by orthogonal design method. The data were analyzed with range analysis and variance analysis. RESULT: To leaf, the optimal medium of callus induction was B5 with 2 mg x L(-1) 2,4-D, 0.5 mg x L(-1) 6-BA, 30 g x L(-1) glucose and 1 g x L(-1) CH; to stem, the optimal one was B5 with2 mg x L(-1) 2,4-D, 1 mg x L(-1) 6-BA, 30 g x L(-1) glucose and 0.5 g x L(-1) CH. In order to obtain higher contents ABPS, to leaf calli, the optimal medium was LS with 1 mg x L(-1) 6-BA, 1 mg x L(-1) TDZ and 30 g x L(-1) sucrose; to stem calli, the optimal one was LS with 1 mg L(-1) 2,4-D, 0.5 mg x L(-1) 6-BA, 1 mg x L(-1) TDZ and 30 g x L(-1) glucose. CONCLUSION: The optimal media of callus induction were established with stems and leaves of A. bidentata as explants and with a view to an industrial production of polysaccharides by tissue and cell culture.