Terpene synthase (TPS) plays important roles in the synthesis of terpenoids which are the main fragrances in Rhododendron flowers. To understand the function of TPS genes in terpenoid metabolism in relation to flower aroma formation, we identified all TPS gene family members in Rhododendron by analyzing its genome database. We then used a transcriptomic approach to analyze the differential gene expression patterns of TPS gene family members in the scented flower Rhododendron fortunei compared to the non-scented flower Rhododendron 'Nova Zembla'. The contents of terpenoid compounds in petals of the above two Rhododendron species at different developmental stages were also measured by using qRT-PCR and head space-solid phase micro-extraction combined with gas chromatography-mass spectrometry. Our results showed that a total of 47 RsTPS members, with individual lengths ranged from 591 to 2 634 bp, were identified in the Rhododendron genome. The number of exons in RsTPS gene ranged from 3 to 12, while the length of each protein encoded ranged from 196 to 877 amino acids. Members of the RsTPS family are mainly distributed in the chloroplast and cytoplasm. Phylogenetic analysis showed that RsTPS genes can be clustered into 5 subgroups. Seven gene family members can be functionally annotated as TPS gene family since they were temporally and spatially expressed as shown in the transcriptome data. Notably, TPS1, TPS10, TPS12 and TPS13 in Rhododendron fortunei were expressed highly in flower buds reached the peak in the full blossoming. Correlation analysis between gene expression levels and terpenoid content indicates that the expression levels of TPS1, TPS4, TPS9, TPS10, TPS12 and TPS13 were positively correlated with the content of terpenoids in the petals of R. fortunei at all flower developmental stages, suggesting that these six genes might be involved in the aroma formation in R. fortunei.
Rhododendron/metabolism* ; Phylogeny ; Terpenes/metabolism* ; Family ; Gene Expression Regulation, Plant

Flavanone 3-hydroxylase (F3H) is a key enzyme in the synthesis of phycocyanidins. In this experiment, the petals of red Rhododendron hybridum Hort. at different developmental stages were used as experimental materials. The R. hybridum flavanone 3-hydroxylase (RhF3H) gene was cloned using reverse transcription PCR (RT-PCR) and rapid-amplification of cDNA ends (RACE) techniques, and bioinformatics analyses were performed. Petal RhF3H gene expression at different developmental stages were analyzed by using quantitative real-time polymerase chain reaction (qRT-PCR). A pET-28a-RhF3H prokaryotic expression vector was constructed for the preparation and purification of RhF3H protein. A pCAMBIA1302-RhF3H overexpression vector was constructed for genetic transformation in Arabidopsis thaliana by Agrobacterium-mediated method. The results showed that the R. hybridum Hort. RhF3H gene is 1 245 bp long, with an open reading frame of 1 092 bp, encoding 363 amino acids. It contains a Fe²⁺ binding motif and a 2-ketoglutarate binding motif of the dioxygenase superfamily. Phylogenetic analysis showed that the R. hybridum RhF3H protein is most closely related to the Vaccinium corymbosum F3H protein. qRT-PCR analysis showed that the expression level of the red R. hybridum RhF3H gene tended to increase and then decrease in the petals at different developmental stages, with the highest expression at middle opening stage. The results of the prokaryotic expression showed that the size of the induced protein of the constructed prokaryotic expression vector pET-28a-RhF3H was about 40 kDa, which was similar to the theoretical value. Transgenic RhF3H Arabidopsis thaliana plants were successfully obtained, and PCR identification and β-glucuronidase (GUS) staining demonstrated that the RhF3H gene was integrated into the genome of A. thaliana plants. qRT-PCR, total flavonoid and anthocyanin contentanalysis showed that RhF3H was significantly higher expressed in the transgenic A. thaliana relative to that of the wild type, and its total flavonoid and anthocyanin content were significantly increased. This study provides a theoretical basis for investigating the function of RhF3H gene, as well as for studying the molecular mechanism of flower color in R. simsiib Planch.
Arabidopsis/metabolism* ; Rhododendron/metabolism* ; Amino Acid Sequence ; Anthocyanins/metabolism* ; Phylogeny ; Flavonoids/metabolism* ; Cloning, Molecular ; Gene Expression Regulation, Plant ; Plant Proteins/metabolism*

This study explores the effect of total flavonoids of Rhododendra simsii(TFR) on middle cerebral artery occlusion(MCAO)-induced cerebral injury in rats and oxygen-glucose deprivation/reoxygenation(OGD/R) injury in PC12 cells and the underlying mechanism. The MCAO method was used to induce focal ischemic cerebral injury in rats. Male SD rats were randomized into sham group, model group, and TFR group. After MCAO, TFR(60 mg·kg~(-1)) was administered for 3 days. The content of tumor necrosis factor-α(TNF-α), interleukin-1(IL-1), and interleukin-6(IL-6) in serum was detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes of brain tissue and cerebral infarction were observed based on hematoxylin and eosin(HE) staining and 2,3,5-triphenyltetrazolium chloride(TTC) staining. RT-qPCR and Western blot were used to detect the mRNA and protein levels of calcium release-activated calcium channel modulator 1(ORAI1), stromal interaction molecule 1(STIM1), stromal intera-ction molecule 2(STIM2), protein kinase B(PKB), and cysteinyl aspartate specific proteinase 3(caspase-3) in brain tissues. The OGD/R method was employed to induce injury in PC12 cells. Cells were randomized into the normal group, model group, gene silencing group, TFR(30 μg·mL~(-1)) group, and TFR(30 μg·mL~(-1))+gene overexpression plasmid group. Intracellular Ca~(2+) concentration and apoptosis rate of PC12 cells were measured by laser scanning confocal microscopy and flow cytometry. The effect of STIM-ORAI-regulated store-operated calcium entry(SOCE) pathway on TFR was explored based on gene silencing and gene overexpression techniques. The results showed that TFR significantly alleviated the histopathological damage of brains in MCAO rats after 3 days of admini-stration, reduced the contents of TNF-α, IL-1, and IL-6 in the serum, down-regulated the expression of ORAI1, STIM1, STIM2, and caspase-3 genes, and up-regulated the expression of PKB gene in brain tissues of MCAO rats. TFR significantly decreased OGD/R induced Ca~(2+) overload and apoptosis in PC12 cells. However, it induced TFR-like effect by ORAI1, STIM1 and STIM2 genes silencing. However, overexpression of these genes significantly blocked the effect of TFR in reducing Ca~(2+) overload and apoptosis in PC12 cells. In summary, in the early stage of focal cerebral ischemia-reperfusion injury and OGD/R-induced injury in PC12 cells TFR attenuates ischemic brain injury by inhibiting the STIM-ORAI-regulated SOCE pathway and reducing Ca~(2+) overload and inflammatory factor expression, and apoptosis.
Animals ; Male ; Rats ; Apoptosis ; Brain Ischemia/metabolism* ; Caspase 3 ; Interleukin-1 ; Interleukin-6 ; Rats, Sprague-Dawley ; Reperfusion Injury/metabolism* ; Tumor Necrosis Factor-alpha/genetics* ; Flavonoids/pharmacology* ; Rhododendron/chemistry*

OBJECTIVETo explore and identify the most potent antihyperglycemic fraction from the ethanol extract of Rhododendron arboreum (R. arboreum) flowers.

METHODSNormal and streptozotocin induced diabetic rats were treated with all four fractions of R. arboreum flowers for short term and with fraction 3 for long term study. On completion of the treatment, a range of indicators were tested including fasting blood glucose, plasma protein, haemoglobin A1C, insulin secretion, body weight, blood lipid profile and carbohydrate metabolism regulating enzymes of liver.

RESULTSIn short term study, the fraction 3 (Active fraction) produced a significant (P<0.000 1) reduction (73.6%) in blood glucose level at a dose of 200 mg/kg after the treatment in the diabetic rats. Administration of active fraction (200 and 400 mg/kg) once daily for 30 d in streptozotocin diabetic rats resulted in a significant (P<0.001 to P<0.000 1) fall in blood glucose level, hemoglobin A1C, serum urea and creatinine with significant but a increase in insulin level similar to standard drug glybenclamide. Further, the active fraction showed antihyperlipidemic activity as evidenced by significant (P<0.001 to P<0.000 1) decreases in serum serum total cholesterol, triglycerides, low density lipoprotein cholesterol and very low density cholesterol levels coupled together with elevation of high density lipoprotein cholesterol in the diabetic rats.

CONCLUSIONSThe active fraction of R. arboreum flowers decreases streptozotocin induced hyperglycemia by promoting insulin secretion and glycolysis and by decreasing gluconeogenesis.

Animals ; Blood Glucose ; drug effects ; Carbohydrate Metabolism ; drug effects ; Diabetes Mellitus, Experimental ; drug therapy ; metabolism ; Disease Models, Animal ; Flowers ; chemistry ; Hypoglycemic Agents ; chemistry ; pharmacology ; Hypolipidemic Agents ; chemistry ; pharmacology ; Lipids ; blood ; Male ; Phytochemicals ; chemistry ; Plant Extracts ; chemistry ; pharmacology ; Rats ; Rhododendron ; chemistry ; Toxicity Tests, Acute