Transcriptional regulation of Lonicera japonica Thunb. during flower development as revealed by comprehensive analysis of transcription factors.

BACKGROUND: Lonicera japonica Thunb. flower has been used for the treatment of various diseases for a long time and attracted many studies on its potential effects. Transcription factors (TFs) regulate extensive biological processes during plant development. As the restricted reports of L. japonica on TFs, our work was carried out to better understand the TFs' regulatory roles under different developmental stages in L. japonica. RESULTS: In this study, 1316 TFs belonging to 52 families were identified from the transcriptomic data, and corresponding expression profiles during the L. japonica flower development were comprehensively analyzed. 917 (69.68%) TFs were differentially expressed. TFs in bHLH, ERF, MYB, bZIP, and NAC families exhibited obviously altered expression during flower growth. Based on the analysis of differentially expressed TFs (DETFs), TFs in MYB, WRKY, NAC and LSD families that involved in phenylpropanoids biosynthesis, senescence processes and antioxidant activity were detected. The expression of MYB114 exhibited a positive correlation with the contents of luteoloside; Positive correlation was observed among the expression of MYC12, chalcone synthase (CHS) and flavonol synthase (FLS), while negative correlation was observed between the expression of MYB44 and the synthases; The expression of LSD1 was highly correlated with the expression of SOD and the total antioxidant capacity, while the expression of LOL1 and LOL2 exhibited a negative correlation with them; Many TFs in NAC and WRKY families may be potentially involved in the senescence process regulated by hormones and reactive oxygen species (ROS). The expression of NAC19, NAC29, and NAC53 exhibited a positive correlation with the contents of ABA and H2O2, while the expression of WRKY53, WRKY54, and WRKY70 exhibited a negative correlation with the contents of JA, SA and ABA. CONCLUSIONS: Our study provided a comprehensive characterization of the expression profiles of TFs during the developmental stages of L. japonica. In addition, we detected the key TFs that may play significant roles in controlling active components biosynthesis, antioxidant activity and flower senescence in L. japonica, thereby providing valuable insights into the molecular networks underlying L. japonica flower development.

Antioxidant and anti-inflammatory activities of Lonicera japonica Thunb. var. sempervillosa Hayata flower bud extracts prepared by water, ethanol and supercritical fluid extraction techniques.

Lonicera japonica Thunberg (LJ) has long been used as an antipyretic, anti-inflammatory and anti-infectious agent in East Asia. The subspecies L. japonica Thunb. var. sempervillosa Hayata (LJv) is a variant that mainly grows in Taiwan. This study examined the antioxidant and anti-inflammatory activities of the extracts from the flower buds of these two species. The extracts were obtained by three extraction methods: water extraction, ethanol extraction, and supercritical-CO2 fluid extraction (SFE). The antioxidant activities of dry LJ (dLJ) extracts were superior to those of LJv extracts. Water extracts possessed higher activities than that prepared by ethanol or SFE. The total polyphenols content, total flavonoids content, and the amount of chlorogenic acid and luteolin-7-O-glucoside were all higher in the water extracts compared to the other two. The SFE extracts of these two species all exhibited excellent anti-inflammatory activities. Although the water and ethanol extracts of dLJ extracts had higher anti-inflammatory activity than that of LJv extracts, the SFE extracts prepared from fresh LJv flower buds (fLJv) exhibited the highest activity among all extracts. The SFE effectively isolates the bioactive components of L. japonica and can obtain the L. japonica extracts with high anti-inflammatory activity.

Validation of the protective effects of Lonicera japonica polysaccharide on lipopolysaccharide-induced learning and memory impairments via regulation of autophagy based on network pharmacology.

BACKGROUND: Learning and memory impairments are important indexes in assessing Alzheimer's disease (AD). Lonicera japonica (L. japonica), a traditional Chinese herbal medicine, inhibits inflammation, but its role in neuroprotection is unclear. Polysaccharide is the main active ingredient in L. japonica. Here, we aimed to validate the effects of L. japonica polysaccharide (LJP) on lipopolysaccharide (LPS)-induced cognitive impairment and the underlying mechanism. METHODS: The Chinese medicine system pharmacology database and analysis platform was used to predict the target of L. japonica; the GeneCards system was used to predict the AD target. We also performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Experiments were performed after bioinformatic analysis for verification. A chronic learning and memory impairment model was established by a single administration of LPS. Learning and memory abilities of Kunming mice were examined after 7 days of induction. The protective effects of LJP on LPS-induced impairment were investigated. Neuronal damage was observed by Nissl staining. Key proteins involved in the autophagy pathway were examined. RESULTS: Bioinformatic analysis showed that there were 151 genes in the intersection of the target and ADrelated genes, and KEGG analysis suggested that these genes may act via multiple pathways. LPS-induced changes in learning and memory in mice were significantly attenuated by LJP. Nissl staining revealed that the neurons in the control group were lost and cellular arrangement was disrupted. LJP alleviated the pathological changes in the neurons of mice. The autophagy pathway was selected to verify the mechanism. ATG5, Beclin 1, Vps34, and LC3 II expression in the LPS group was significantly increased, and it was further increased in the LJP group. CONCLUSIONS: LJP improved behavioral changes and neuronal loss associated with LPS-induced learning and memory impairments. The underlying mechanism may be related to the regulation of the autophagy pathways.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of the effector-interaction domain of the resistance protein RGA5-A from Oryza sativa L. japonica.

RGA5-A, a component of the Pia resistance-protein complex (RGA4/RGA5-A) from Oryza sativa L. japonica, has the ability to interact physically with the effector protein AVR-Pia from Magnaporthe oryzae via its effector-interaction domain RGA5-A_S. The interaction between RGA5-A and AVR-Pia relieves the repression of RGA4, leading to AVR-independent cell death by the freed RGA4. To further understand the details of this interaction, the effector-interaction domain RGA5-A_S was expressed in Escherichia coli and purified to homogeneity. The purified recombinant protein His-RGA5-A_S was successfully crystallized using the sitting-drop vapour-diffusion method. A single crystal obtained using 0.2 M ammonium citrate, 25%(w/v) PEG 3350 diffracted to 2.43 Å resolution. It belonged to space group P4122 or P4322, with unit-cell parameters a = b = 55.2, c = 78.2 Å, α = ß = γ = 90°.

Crystallization and preliminary X-ray crystallographic studies of dehydroascorbate reductase (DHAR) from Oryza sativa L. japonica.

Dehydroascorbate reductase from Oryza sativa L. japonica (OsDHAR), a key enzyme in the regeneration of vitamin C, maintains reduced pools of ascorbic acid to detoxify reactive oxygen species. In previous studies, the overexpression of OsDHAR in transgenic rice increased grain yield and biomass as well as the amount of ascorbate, suggesting that ascorbate levels are directly associated with crop production in rice. Hence, it has been speculated that the increased level of antioxidants generated by OsDHAR protects rice from oxidative damage and increases the yield of rice grains. However, the crystal structure and detailed mechanisms of this important enzyme need to be further elucidated. In this study, recombinant OsDHAR protein was purified and crystallized using the sitting-drop vapour-diffusion method at pH 8.0 and 298 K. Plate-shaped crystals were obtained using 0.15 M potassium bromide, 30%(w/v) PEG MME 2000 as a precipitant, and the crystals diffracted to a resolution of 1.9 Šon beamline 5C at the Pohang Accelerator Laboratory. The X-ray diffraction data indicated that the crystal contained one OsDHAR molecule in the asymmetric unit and belonged to space group P21 with unit-cell parameters a=47.03, b=48.38, c=51.83 Å, ß=107.41°.

Purification, characterization and preliminary X-ray crystallographic studies of monodehydroascorbate reductase from Oryza sativa L. japonica.

Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) is a key enzyme in the reactive oxygen species (ROS) detoxification system of plants. The participation of MDHAR in ascorbate (AsA) recycling in the ascorbate-glutathione cycle is important in the acquired tolerance of crop plants to abiotic environmental stresses. Thus, MDHAR represents a strategic target protein for the improvement of crop yields. Although physiological studies have intensively characterized MDHAR, a structure-based functional analysis is not available. Here, a cytosolic MDHAR (OsMDHAR) derived from Oryza sativa L. japonica was expressed using Escherichia coli strain NiCo21 (DE3) and purified. The purified OsMDHAR showed specific enzyme activity (approximately 380 U per milligram of protein) and was crystallized using the hanging-drop vapour-diffusion method at pH 8.0 and 298 K. The crystal diffracted to 1.9 Šresolution and contained one molecule in the asymmetric unit (the Matthews coefficient VM is 1.98 Å(3) Da(-1), corresponding to a solvent content of 38.06%) in space group P41212 with unit-cell parameters a = b = 81.89, c = 120.4 Å. The phase of the OsMDHAR structure was resolved by the molecular-replacement method using a ferredoxin reductase from Acidovorax sp. strain KKS102 (PDB entry 4h4q) as a model.