Isolation, Identification, Phytochemical Characterization, and In Vitro Anti-Inflammatory Property of an Endophytic Trametes versicolor CL-1 Isolated from Rosa roxburghii.

This study explores the anti-inflammatory potential of an endophytic fungus, Trametes versicolor CL-1, isolated from the fruit tissues of Rosa roxburghii. Morphological and molecular analyses confirmed the identity of CL-1. An ethyl acetate extract (CL-E) from its fermentation broth was subjected to UPLC-HRMS and GNPS molecular networking. The analysis revealed a diverse array of secondary metabolites, including 11 terpenes, 7 flavonoids, 10 cinnamic acid derivatives, 6 oligopeptides, and 9 fatty acids, as verified by LC-MS/MS. Notably, CL-E exhibited significant in vitro anti-inflammatory activity in RAW264.7 cells. Furthermore,  molecular docking studies predicted favorable binding interactions of key compounds 1 within CL-E with the NLRP3 inflammasome (PDB ID: 6NPY). These findings suggest T. versicolor CL-1 as a promising source of natural anti-inflammatory agents and unveil R. roxburghii as a potential reservoir for discovering novel bioactive metabolites.

Fine-Scale analysis of both wild and cultivated horned galls provides insight into their quality differentiation.

BACKGROUND: Galla chinensis is a traditional Chinese medicine (TCM) produced due to the interaction between the Fordinae aphids and the Rhus plant species. Horned galls with high tannin content are the most widely cultivated gall type, and Wufeng county of Hubei province in China is the center of cultivation. However, long-term artificial cultivation and domestication of horned galls to meet the increasing production demand have led to quality degradation. Understanding the reasons underlying quality degradation is urgent for horned gall production and application. The present study used a combination of metabolic, genetic, and ecological analyses to investigate the quality and genetic differentiation of the horned galls under long-term domestication as well as the potential relationships between them. RESULTS: Analysis of gallic acid content and other three phenotypic traits (fresh weight, gall size, and wall thickness) revealed quality differentiation of horned galls collected from five locations in Wufeng, in which the cultivated samples from Wang Jiaping (WJP) showed the highest degradation. Genetic differentiation between the cultivated and wild Rhus chinensis trees in WJP, and between WJP and the other populations was detected based on SSR molecular markers, however, no significant difference in genetic structure was seen for the aphid populations. Among the various ecological factors examined, temperature was identified as the primary one affecting the quality of horned galls. CONCLUSIONS: Both genetic and ecological factors caused quality differentiation of horned galls. The collection of diverse germplasm of host trees and aphids will help reduce the quality degradation of horned galls in Wufeng.

Neuroprotection of Kaji-Ichigoside F1 via the BDNF/Akt/mTOR Signaling Pathways against NMDA-Induced Neurotoxicity.

Kaji-ichigoside F1 (KF1), a natural oleanane-type triterpenoid saponin, is the main active constituent from Rosa roxburghii. In the southwest regions of China, particularly in Guizhou Province, this plant was used as a Miao ethnic medicine to prevent and treat dyspepsia, dysentery, hypoimmunity, and neurasthenia. In the present study, the neuroprotective effect of KF1 was evaluated against N-methyl-D-aspartate (NMDA)-induced neurotoxicity in vivo and in vitro. An NMDA-induced PC12 cell neurotoxicity assay showed that KF1 effectively improved cellular viability, inhibited the release of lactate dehydrogenase (LDH), and reduced cell apoptosis. Furthermore, KF1-treated NMDA-induced excitotoxicity mice displayed a remarkable capacity for improving spatial learning memory in the Y-maze and Morris water maze tests. In addition, KF1 increased the levels of the neurotransmitters 5-hydroxytryptamine, dopamine, and monoamine oxidase and reduced the calcium ion concentration in the hippocampus of mice. Hematoxylin and eosin and Nissl staining indicated that KF1 effectively reduced the impairment of neurons. Furthermore, Western blot assays showed that KF1 decreased NMDAR1 expression. In contrast, the NMDAR2B (NR2B), glutamate receptor (AMPA), TrkB, protein kinase B (AKT), mammalian target of rapamycin (mTOR), PSD95, and synapsin 1 were upregulated in NMDA-induced PC12 cells and an animal model. These results suggest that KF1 has a remarkable protective effect against NMDA-induced neurotoxicity, which is directly related to the regulation of the NMDA receptor and the activation of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) and BDNF/AKT/mTOR signaling pathways.

De novo transcriptome sequencing and gene expression profiling of Magnolia wufengensis in response to cold stress.

BACKGROUND: Magnolia wufengensis is a new species of Magnolia L. and has considerable ornamental and economic value due to its unique characteristics. However, because of its characteristic of poor low temperature resistance, M. wufengensis is hardly popularization and application in the north of China. Furthermore, the mechanisms of gene regulation and signaling pathways involved in the cold-stress response remained unclear in this species. In order to solve the above-mentioned problems, we performed de novo transcriptome assembly and compared the gene expression under the natural (25 °C) and cold (4 °C) conditions for M. wufengensis seedlings. RESULTS: More than 46 million high-quality clean reads were produced from six samples (RNA was extracted from the leaves) and were used for performing de novo transcriptome assembly. A total of 59,764 non-redundant unigenes with an average length of 899 bp (N50 = 1,110) were generated. Among these unigenes, 31,038 unigenes exhibited significant sequence similarity to known genes, as determined by BLASTx searches (E-value ≤1.0E-05) against the Nr, SwissProt, String, GO, KEGG, and Cluster of COG databases. Based on a comparative transcriptome analysis, 3,910 unigenes were significantly differentially expressed (false discovery rate [FDR] < 0.05 and |log2FC (CT/CK)| ≥ 1) in the cold-treated samples, and 2,616 and 1,294 unigenes were up- and down-regulated by cold stress, respectively. Analysis of the expression patterns of 16 differentially expressed genes (DEGs) by quantitative real-time RT-PCR (qRT-PCR) confirmed the accuracy of the RNA-Seq results. Gene Ontology and KEGG pathway functional enrichment analyses allowed us to better understand these differentially expressed unigenes. The most significant transcriptomic changes observed under cold stress were related to plant hormone and signal transduction pathways, primary and secondary metabolism, and photosynthesis. In addition, 113 transcription factors, including members of the AP2-EREBP, bHLH, WRKY, MYB, NAC, HSF, and bZIP families, were identified as cold responsive. CONCLUSION: We generated a genome-wide transcript profile of M. wufengensis and a de novo-assembled transcriptome that can be used to analyze genes involved in biological processes. In this study, we provide the first report of transcriptome sequencing of cold-stressed M. wufengensis. Our findings provide important clues not only for understanding the molecular mechanisms of cold stress in plants but also for introducing cold hardiness into M. wufengensis.

Icariin Delays Brain Aging in Senescence-Accelerated Mouse Prone 8 (SAMP8) Model via Inhibiting Autophagy.

Icariin (ICA), a major flavonoid extracted from the Chinese tonic herb Epimedium, exerts beneficial effects in a variety of age-dependent diseases, such as Alzheimer's disease (AD). However, the antiaging mechanisms remain unclear. The senescence-accelerated mouse-prone 8 (SAMP8) model has been used to study age-related neurodegenerative changes associated with aging and the pathogenesis of AD. Hence, the current study was designed to examine the effect of ICA on age-related cognitive decline in SAMP8 mice and explore the role of autophagy in the ICA-mediated neuroprotection. SAMP8 mice were administered with ICA starting at 5 months of age, and the treatment lasted for 3 consecutive months. Morris water maze was used to evaluate cognitive function. The senescence-associated ß-galactosidase staining was used to determine the number of senescence cells. The neuronal morphologic changes were examined via Nissl staining. The hippocampal neuronal ultrastructure was examined by transmission electron microscopy. The expression of autophagy protein was examined by Western blot. ICA-treated SAMP8 mice exhibited a robust improvement in spatial learning and memory function. Meanwhile, ICA reduced the number of senescence cells in the brains of SAMP8 mice, inhibited neuronal loss, and reversed neuronal structural changes in the hippocampi of SAMP8 mice. Moreover, ICA treatment also decreased the formation of autophagosomes in the hippocampus of SAMP8 mice, and reduced the expression of autophagy-related proteins LC3-II and p62. These results demonstrate that ICA possesses the ability to delay brain aging in SAMP8 mice, and the mechanisms are possibly mediated through the regulation of autophagy.

Trichoderma harzianum mitigates salt stress in cucumber via multiple responses.

Trichoderma harzianum T-soybean plays an important role in controlling soybean root rot disease. However, the mechanism by which it improves plant tolerance to salt stress is not clear. In this study, we investigated the possible mechanism of T-soybean in mitigating the damage caused by salt stress in Cucumis sativus L plants. Our results suggest that T-soybean improved salt tolerance of cucumber seedlings by affecting the antioxidant enzymes including peroxidase (POD) (EC 1.11.1.6), polyphenol oxidase (PPO) (EC 1.14.18.1), phenylalanine ammonia-lyase (PAL) (EC 4.3.1.5), catalase (CAT) (EC 1.11.1.6), superoxide dismutase (SOD) (EC 1.15.1.1), ascorbate peroxidase (APX) (EC 1.11.1.11), and glutathione reductase (GR) (EC 1.6.4.2), by increasing the levels of proline, soluble sugars, soluble protein, ascorbic acid (AsA) and chlorophyll as well as improving root activity. Treatment with T-soybean improved the ratio of glutathione (GSH)/oxidized glutathione (GSSG) and AsA/dehydroascorbate (DHA), and up-regulated the expression of CsAPX and CsGR genes involved in the AsA-GSH cycle. In addition, treatment with T-soybean increased the K+ content and K+/Na+ ratio while decreased the Na+ concentration and ethylene level. In summary, the improved salt tolerance of cucumber plants may be due to multiple mechanisms of T-soybean, such as the increase in reactive oxygen species (ROS) scavenging, as well as maintaining osmotic balance and metabolic homeostasis under salt stress.

Fermented Gastrodia elata Bl. Alleviates Cognitive Deficits by Regulating Neurotransmitters and Gut Microbiota in D-Gal/AlCl3-Induced Alzheimer's Disease-like Mice.

Alzheimer's disease (AD) is a common neurological disease with recognition ability loss symptoms and a major contributor to dementia cases worldwide. Gastrodia elata Bl. (GE), a food of medicine-food homology, has been reported to have a mitigating effect on memory and learning ability decline. However, the effect of GE fermented by Lactobacillus plantarum, Acetobacter pasteurianus, and Saccharomyces (FGE) on alleviating cognitive deficits in AD was not studied. Mice were randomly divided into six groups, control, model, donepezil, low, medium, and high doses of FGE, and D-Galactose/Aluminum chloride (D-Gal/AlCl3) was used to establish an AD-like mouse model. The results indicated that FGE could improve the production of neurotransmitters and relieve oxidative stress damage in AD-like mice, which was evidenced by the declined levels of amyloid-ß (Aß), Tau, P-Tau, acetylcholinesterase (AchE), and malondialdehyde (MDA), and increased acetylcholine (Ach), choline acetyltransferase (ChAT), and superoxide dismutase (SOD) levels in brain tissue. Notably, FGE could enhance the richness of the gut microbiota, especially for beneficial bacteria such as Lachnospira and Lactobacillus. Non-target metabolomics results indicated that FGE could affect neurotransmitter levels by regulating amino acid metabolic pathways to improve AD symptoms. The FGE possessed an ameliorative effect on AD by regulating neurotransmitters, oxidative stress levels, and gut microbiota and could be considered a good candidate for ameliorating AD.

Natural product Kaji-ichigoside F1 exhibits rapid antidepression via activating the AMPA-BDNF-mTOR pathway and inhibiting the NMDAR-CaMKIIα pathway.

BACKGROUND: Depression is a common and recurrent neuropsychiatric disorder. Recent studies have shown that the N-methyl-d-aspartate (NMDA) receptor (NMDAR) is involved in the pathophysiology of depression. Previous studies have found that Kaji-ichigoside F1 (KF1) has a protective effect against NMDA-induced neurotoxicity. However, the antidepressant mechanism of KF1 has not been confirmed yet. PURPOSE: In the present study, we aimed to evaluate the rapid antidepressant activity of KF1 and explore the underlying mechanism. STUDY DESIGN: First, we explored the effect of KF1 on NMDA-induced hippocampal neurons and the underlying mechanism. Second, depression was induced in C57BL/6 mice via chronic unpredictable mild stress (CUMS), and the immediate and persistent depression-like behavior was evaluated using the forced swimming test (FST) after a single administration of KF1. Third, the contributions of NMDA signaling to the antidepressant effect of KF1 were investigated using pharmacological interventions. Fourth, CUMS mice were treated with KF1 for 21 days, and then their depression-like behaviors and the underlying mechanism were further explored. METHODS: The FST was used to evaluate immediate and persistent depression-like behavior after a single administration of KF1 with or without NMDA pretreatment. The effect of KF1 on depressive-like behavior was investigated in CUMS mice by treating them with KF1 once daily for 21 days through the sucrose preference test, FST, open field test, and tail suspension test. Then, the effects of KF1 on the morphology and molecular and functional phenotypes of primary neuronal cells and hippocampus of mice were investigated by hematoxylin-eosin staining, Nissl staining, propidium iodide staining, TUNEL staining, Ca2+ imaging, JC-1 staining, ELISA, immunofluorescence analysis, RT-PCR, and Western blot. RESULTS: KF1 could effectively improve cellular viability, reduce apoptosis, inhibit the release of LDH and Ca2+, and increase the mitochondrial membrane potential and the number of dendritic spines numbers in hippocampal neurons. Moreover, behavioral tests showed that KF1 exerted acute and sustained antidepressant-like effects by reducing Glu-levels and ameliorating neuronal damage in the hippocampus. Additionally, in vivo and in vitro experiments revealed that PSD95, Syn1, α-amino-3­hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and brain-derived neurotrophic factor (BDNF) were upregulated at the protein level, and BDNF and AMPA were upregulated at the mRNA level. NR1 and NR2A showed the opposite trend. CONCLUSION: These results confirm that KF1 exerts rapid antidepressant effects mainly by activating the AMPA-BDNF-mTOR pathway and inhibiting the NMDAR-CaMKIIα pathway. This study serves as a new reference for discovering rapid antidepressants.

Inhibitory effect of Incarvillea diffusa Royle extract in the formation of calcium oxalate nephrolithiasis by regulating ROS-induced Nrf2/HO-1 pathway in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Calcium oxalate (CaOx) kidney stones are widely acknowledged as the most prevalent type of urinary stones, with high incidence and recurrence rates. Incarvillea diffusa Royle (ID) is a traditionally used medicinal herb in the Miao Minzu of Guizhou province, China, for treating urolithiasis. However, the active components and the underlying mechanism of its pharmacodynamic effects remain unclear. AIM OF THE STUDY: This study aimed to investigate the potential inhibitory effect of the active component of ID on the formation of CaOx nephrolithiasis and elucidate the underlying mechanism. MATERIALS AND METHODS: In vivo, a CaOx kidney stone model was induced in Sprague-Dawley (SD) rats using an ethylene glycol and ammonium chloride protocol for four weeks. Forty-eight male SD rats were randomly assigned to 6 groups (n = 8): blank group, model group, apocynin group, and low, medium, and high dose of ID's active component (IDW) groups. After three weeks of administration, rat urine, serum, and kidney tissues were collected. Renal tissue damage and crystallization, Ox, BUN, Ca2+, CRE, GSH, MDA, SOD contents, and levels of IL-1ß, IL-18, MCP-1, caspase-1, IL-6, and TNF-α in urine, serum, and kidney tissue were assessed using HE staining and relevant assay kits, respectively. Protein expression of Nrf2, HO-1, p38, p65, and Toll-4 in kidney tissues was quantified via Western blot. The antioxidant capacities of major compounds were evaluated through DPPH, O2·-, and ·OH radical scavenging assays, along with their effects on intracellular ROS production in CaOx-induced HK-2 cells. RESULTS: We found that IDW could significantly reduce the levels of CRE, GSH, MDA, Ox, and BUN, and enhancing SOD activity. Moreover, it could inhibit the secretion of TNF-α, IL-1ß, IL-18, MCP-1, caspase-1, and decreased protein expression of Nrf2, HO-1, p38, p65, and Toll-4 in renal tissue. Three major compounds isolated from IDW exhibited promising antioxidant activities and inhibited intracellular ROS production in CaOx-induced HK-2 cells. CONCLUSIONS: IDW facilitated the excretion of supersaturated Ca2+ and decreased the production of Ox, BUN in SD rat urine, and mitigated renal tissue damage by regulating Nrf2/HO-1 signaling pathway. Importantly, the three major compounds identified as active components of IDW contributed to the inhibition of CaOx nephrolithiasis formation. Overall, IDW holds significant potential for treating CaOx nephrolithiasis.

Anti-depressant-like effect of fermented Gastrodia elata Bl. by regulating monoamine levels and BDNF/NMDAR pathways in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Gastrodia elata Blume (GE) is a Chinese medicinal herb commonly used to treat central nervous system-related diseases, including headaches, dizziness, epilepsy, numbness of the limbs and depression. AIM OF THE STUDY: Microbial-based fermentation has been successfully used to increase the extract efficiency of medicinal herbs in recent years. However, no study has hitherto explored the anti-depressant-like effect of GE processed by microorganisms. Herein, this subject aimed to clarify the anti-depressant-like effect of fermented Gastrodia elata Bl. (FGE) and its active chemical constituents. MATERIALS AND METHODS: The chronic unpredictable mild stress (CUMS) model, a well-established animal model of depression, was induced in Kunming (KM) mice. The mice were administrated with FGE for 3 weeks. The sucrose preference test (SPT), open field test (OFT) and tail suspension test (TST) were conducted. Moreover, the levels of serotonin (5-HT) and dopamine (DA) in brain tissue homogenates, the concentration of Ca2+ and the activity of MAO in serum, H&E and Nissl staining in the hippocampus, and the hippocampus protein expressions of BDNF, NMDAR1, NMDAR2A and NMDAR2B relevant to depression were detected. Furthermore, chemical constituents of FGE were further isolated, and the protective activity of the obtained compounds against NMDA-induced PC-12 cell damage was assessed. RESULTS: FGE could alleviate the depression state in CUMS-induced mice and reduce apoptosis of neuronal cells in the hippocampus. Furthermore, FGE could improve the contents of 5-HT, DA and decrease the concentration of Ca2+ and MAO activity in brain tissue and serum compared with the control group. It could reverse the decreased expression of BDNF, NMDAR2A and NMDAR2B and increase NMDAR1 protein expression. Investigation of the active constituents from FGE yielded two new compounds, (4-(((4-ethoxybenzyl) oxy)methyl)-phenol 1 and 3-((4-hydroxy benzyl)oxy)propane-1,2-diol) 2, with twelve known compounds (3-14). The compounds (3-((4-hydroxybenzyl)oxy)propane-1,2-diol 2, 4, 4'-dihydroxyd iphenyl methane 3, and bungein A 4) protected against NMDA-induced PC-12 cells damage. CONCLUSION: This study demonstrated that FGE could improve the depressive behavior of CUMS-induced mice and exert a protective effect on nerve cells in the brain. Importantly, compounds 2-4 are the active components of FGE. Overall, the above findings suggest that FGE has huge prospects for application in treating depression-related diseases.

Cinnamigones A-C, three highly oxidized guaiane-type sesquiterpenes with neuroprotective activity from Cinnamomum migao.

Cinnamigones A-C, three undescribed highly oxidized guaiane-type sesquiterpenes were isolated from the fruits of Cinnamomum migao. Cinnamigone A (1), structurally artemisinin-like, is a natural 1,2,4-trioxane caged endoperoxide with an unprecedented tetracyclic 6/6/7/5 ring system. Compounds 2-3 are classic guaiane sesquiterpene featuring different epoxy units. Guaiol (4) is considered to be the precursor of 1-3 in the biosynthesis pathway hypothesis. The planar structures and configurations of cinnamigones A-C were elucidated by spectral analysis, HRESIMS, X-ray crystallography and ECD calculations. Evaluation of the neuroprotective activity of 1-3 on N-methyl-ᴅaspartate (NMDA) toxicity was demonstrated that compounds 1-2 exhibited moderate neuroprotective activity against NMDA-induced neurotoxicity.

A high-affinity anti-salbutamol monoclonal antibody: key to a robust lateral-flow immunochromatographic assay.

Among the components that make up a lateral-flow immunochromatographic assay (ICA), antibody is the key. In this paper, salbutamol (SAL) as a model analyte was meticulously designed to prepare immunogen and coating antigen in distinctly different ways. Four hybridoma cell lines were prepared and identified. Among them, C9 had highest affinity, best dose-response behavior, lowest limit of detection, and highest specificity and was chosen to be labeled with colloidal gold as the detector reagent and applied on the conjugate pad. Goat anti-mouse antibody and SAL-BSA conjugate were sprayed on a nitrocellulose membrane as test line and control line, respectively. Under the optimized conditions, the ICA strip was constructed based on a binding inhibition format. Color intensity on the test line was visually distinguishable from that of the negative sample within 5 min, with the visual detection limit of 1 ngml(-1) in phosphate-buffered saline. Cross-reactions with other ß-agonists were not found (<1%). The results from ICA were in a good agreement with those obtained by enzyme-linked immunosorbent assay. The developed ICA has potential as a useful on-site screening tool for SAL in swine urine.

Norbisabolane-type sesquiterpenoid derivatives, benzofuran lignans and a phenolic glycoside from the roots of Glochidion wilsonii Hutch.

Three highly oxygenated norbisabolane sesquiterpenoid glycosides (glochiwilsonosides A-C), five benzofuran lignans (glochiwilsonises A-E) and a phenolic glycoside (glochiwilsophe-noside), together with forty-one known compounds, were isolated from the roots of Glochidion wilsonii Hutch. The chemical structures of the compounds were identified by spectroscopic methods and previous literature data. Glochiwilsonoside A displayed anti-proliferative activity on A-549 and RAW 264.7 cell lines with an IC50 value of 34.5 ± 0.9 µM and CC50 value of 16.0 ± 0.9 µM, respectively. Twenty-three known compounds were reported from the genus Glochidion for the first time, and the chemotaxonomic characteristics of the isolated compounds were also summarized. The bisabolane/norbisabolane-type sesqui-terpenoid derivatives could be used as chemotaxonomic markers for G. wilsonii.

MawuAP1 promotes flowering and fruit development in the basal angiosperm Magnolia wufengensis (Magnoliaceae).

The APETALA1/SQUAMOSA (AP1/SQUA)-like genes of flowering plants play crucial roles in the development processes of floral meristems, sepals, petals and fruits. Although many of the AP1/SQUA-like genes have been characterized in angiosperms, few have been identified in basal angiosperm taxa. Therefore, the functional evolution of the AP1/SQUA subfamily is still unclear. We characterized an AP1 homolog, MawuAP1, from Magnolia wufengensis that is an ornamental woody plant belonging to the basal angiosperms. Gene sequence and phylogenetic analyses suggested that MawuAP1 was clustered with the FUL-like homologous genes of basal angiosperms and had FUL motif and paleoAP1 motif domain, but it did not have the euAP1 motif domain of core eudicots. Expression pattern analysis showed that MawuAP1 was highly expressed in vegetative and floral organs, particularly in the early stage of flower bud development and pre-anthesis. Protein-protein interaction pattern analysis revealed that MawuAP1 has interaction with an A-class gene (MawuAP1), C-class gene (MawuAG-1) and E-class gene (MawuAGL9) of the MADS-box family genes. Ectopic expression in Arabidopsis thaliana indicated that MawuAP1 could significantly promote flowering and fruit development, but it could not restore the sepal and petal formation of ap1 mutants. These results demonstrated that there are functional differences in the specification of sepal and petal floral organs and development of fruits among the AP1/SQUA-like genes, and functional conservation in the regulation of floral meristem. These findings provide strong evidence for the important functions of MawuAP1 in floral meristem determination, promoting flowering and fruit development, and further highlight the importance of AP1/SQUA subfamily in biological evolution and diversity.

Conservation and divergence of ancestral AGAMOUS/SEEDSTICK subfamily genes from the basal angiosperm Magnolia wufengensis.

AGAMOUS/SEEDSTICK (AG/STK) subfamily genes play crucial roles in the reproductive development of plants. However, most of our current knowledge of AG/STK subfamily genes is restricted to core eudicots and grasses, and the knowledge of ancestral exon-intron structures, expression patterns, protein-protein interaction patterns and functions of AG/STK subfamily genes remains unclear. To determine these, we isolated AG/STK subfamily genes (MawuAG1, MawuAG2 and MawuSTK) from a woody basal angiosperm Magnolia wufengensis (Magnoliaceae). MawuSTK arose from the gene duplication event occurring before the diversification of extant angiosperms, and MawuAG1 and MawuAG2 may result from a gene duplication event occurring before the divergence of Magnoliaceae and Lauraceae. Gene duplication led to apparent diversification in their expression and interaction patterns. It revealed that expression in both stamens and carpels likely represents the ancestral expression profiles of AG lineage genes, and expression of STK-like genes in stamens may have been lost soon after the appearance of the STK lineage. Moreover, AG/STK subfamily proteins may have immediately established interactions with the SEPALLATA (SEP) subfamily proteins following the emergence of the SEP subfamily; however, their interactions with the APETALA1/FRUITFULL subfamily proteins or themselves differ from those found in monocots and basal and core eudicots. MawuAG1 plays highly conserved roles in the determinacy of stamen, carpel and ovule identity, while gene duplication contributed to the functional diversification of MawuAG2 and MawuSTK. In addition, we investigated the evolutionary history of exon-intron structural changes of the AG/STK subfamily, and a novel splice-acceptor mode (GUU-AU) and the convergent evolution of N-terminal extension in the euAG and PLE subclades were revealed for the first time. These results further advance our understanding of ancestral AG/STK subfamily genes in terms of phylogeny, exon-intron structures, expression and interaction patterns, and functions, and provide strong evidence for the significance of gene duplication in the expansion and evolution of the AG/STK subfamily.

Comparative transcriptome analysis highlights the hormone effects on somatic embryogenesis in Catalpa bungei.

KEY MESSAGE: The major pathways and key events related to somatic embryo development in Catalpa bungei were illustrated by deep analysis of DEGs and quantification of hormone contents. Catalpa bungei C.A. Meyer is a valuable timber species, known as "The king of wood" in China. Due to the low propagation rate, somatic embryogenesis-based rapid propagation can regenerate a large number of new plants in a very short period of time and thus has great commercial value for this timber species. However, the mechanisms of somatic embryogenesis in C. bungei remain largely unclear so far. In our previous study, we established the vegetative propagation system in C. bungei using immature zygotic embryo as explants. Here, we further compared the transcriptional profiles and hormones contents between the embryogenic callus (EC) and non-embryogenic callus (NEC). RNA-seq analysis showed a total assembly of 73038 unigenes, and identified 12310 differentially expressed genes (DEGs) between EC and NEC. Also, six DEGs were chosen to verify the authenticity of the transcriptome sequencing results by qRT-PCR. Moreover, by using LC-MS approaches, we quantified various plant hormone contents and found that auxin and ABA were dramatically higher in EC than those in NEC. Accordingly, DEGs were enriched in plant hormone signaling pathways. Taken together, we highlight the hormone effects on somatic embryogenesis in a tree species, C. bungei. The use of certain genes as markers of embryogenesis induction in C. bungei regeneration process will provide new tools to pre-screen genotypes or tissue culture hormone combinations suitable for somatic embryo production. Our results provide theoretical references for the somatic embryogenesis mechanism and experimental bases for breeding and rapid propagation of C. bungei.

Gene duplication led to divergence of expression patterns, protein-protein interaction patterns and floral development functions of AGL6-like genes in the basal angiosperm Magnolia wufengensis (Magnoliaceae).

The MADS-box family genes play critical roles in the regulation of growth and development of flowering plants. AGAMOUS-LIKE 6 (AGL6)-like genes are one of the most enigmatic subfamilies of the MADS-box family because of highly variable expression patterns and ambiguous functions, which have long puzzled researchers. A lot of AGL6 homologs have been identified from gymnosperms and angiosperms. However, only a few have been characterized, especially for basal angiosperm taxa. Magnolia wufengensis is a woody basal angiosperm from the family Magnoliaceae. In the current study, the phylogenesis, expression and protein-protein interaction (PPI) patterns, and functions of two AGL6 homologs from M. wufengensis, MawuAGL6-1 and MawuAGL6-2, were analyzed. Phylogenetic analysis indicated that the two AGL6 duplicates may have arisen by gene duplication before the divergence of Magnoliaceae and Lauraceae, with the diversification of their expression and PPI patterns after gene duplication. Functional analysis revealed that, in addition to common functions in accelerating flowering, MawuAGL6-1 might be responsible for flower meristem determinacy, while MawuAGL6-2 is preferentially recruited to regulate tepal morphogenesis. These findings further advance our understanding of the evolution of phylogenesis, expression, interaction and functions of AGL6 lineage genes from basal angiosperms, as well as the entire AGL6 lineage genes, and the significance of AGL6 lineage genes in the evolution and biological diversity.

Isolation and Characterization of AGAMOUS-Like Genes Associated With Double-Flower Morphogenesis in Kerria japonica (Rosaceae).

Double-flower phenotype is more popular and attractive in garden and ornamental plants. There is great interest in exploring the molecular mechanisms underlying the double-flower formation for further breeding and selection. Kerria japonica, a commercial ornamental shrub of the Rosaceae family, is considered an excellent system to determine the mechanisms of morphological alterations, because it naturally has a single-flower form and double-flower variant with homeotic conversion of stamens into petals and carpels into leaf-like carpels. In this study, Sf-KjAG (AGAMOUS homolog of single-flower K. japonica) and Df-KjAG (AGAMOUS homolog of double-flower K. japonica) were isolated and characterized as two AGAMOUS (AG) homologs that occur strictly in single- and double-flower K. japonica, respectively. Our sequence comparison showed that Df-KjAG is derived from ectopic splicing with the insertion of a 2411 bp transposon-like fragment, which might disrupt mRNA accumulation and protein function, into intron 1. Ectopic expression analysis in Arabidopsis revealed that Sf-KjAG is highly conserved in specifying carpel and stamen identities. However, Df-KjAG did not show any putative C-class function in floral development. Moreover, yeast-two-hybrid assays showed that Sf-KjAG can interact with KjAGL2, KjAGL9, and KjAP1, whereas Df-KjAG has lost interactions with these floral identity genes. In addition, loss-of-function of Df-KjAG affected not only its own expression, but also that of other putative floral organ identity genes such as KjAGL2, KjAGL9, KjAP1, KjAP2, KjAP3, and KjPI. In conclusion, our findings suggest that double-flower formation in K. japonica can be attributed to Df-KjAG, which appears to be a mutant produced by the insertion of a transposon-like fragment in the normal AG homolog (Sf-KjAG) of single-flower K. japonica. Highlights:Sf-KjAG and Df-KjAG are different variations only distinguished by a transposon-like fragment insertion which lead to the evolutionary transformation from single-flower to double-flowers morphogenesis in Kerria japonica.

Isolation and Functional Analysis of PISTILLATA Homolog From Magnolia wufengensis.

PISTILLATA (PI) homologs are crucial regulators of flower development in angiosperms. In this study, we isolated the MAwuPI homolog from Magnolia wufengensis, a basal angiosperm belonging to the Magnoliaceae. Molecular phylogenetic analysis suggested that MAwuPI was grouped into the PI/GLO lineages of B-class MADS-box gene with the distinctive PI motif. Further expression profiling analysis showed that MAwuPI was expressed in tepals and stamens but not in juvenile leaves and carpels, similar to the spatial expression pattern of AtPI in Arabidopsis. Interestingly, MAwuPI had higher expression level in inner-tepals than in outer-tepals, whereas the M. wufengensis flower is homochlamydeous. Moreover, ectopic expression of MAwuPI in Arabidopsis pi-1 mutant emerged filament-like structures but had no obvious petals, suggesting a partial phenotypic recovery of pi-1 mutant. The features of MAwuPI in the expression pattern and gene function improved our acknowledgment of B-class genes in M. wufengensis, and contributed to the clarification of M. wufengensis evolution status and relations with other sibling species in molecular perspective.

Floral organ MADS-box genes in Cercidiphyllum japonicum (Cercidiphyllaceae): Implications for systematic evolution and bracts definition.

The dioecious relic Cercidiphyllum japonicum is one of two species of the sole genus Cercidiphyllum, with a tight inflorescence lacking an apparent perianth structure. In addition, its systematic place has been much debated and, so far researches have mainly focused on its morphology and chloroplast genes. In our investigation, we identified 10 floral organ identity genes, including four A-class, three B-class, two C-class and one D-class. Phylogenetic analyses showed that all ten genes are grouped with Saxifragales plants, which confirmed the phylogenetic place of C. japonicum. Expression patterns of those genes were examined by quantitative reverse transcriptase PCR, with some variations that did not completely coincide with the ABCDE model, suggesting some subfunctionalization. As well, our research supported the idea that thebract actually is perianth according to our morphological and molecular analyses in Cercidiphyllum japonicum.