The complete chloroplast genome of Crepis rigescens (Cichorioideae), a traditional Chinese medicinal plant.

Crepis rigescens, Diels 1921 is a traditional Chinese medicinal plant of Cichorioideae, which contains many chemicals, such as friedelin, ß-sitosterol, stigmasterol, chlorogenic acid, and flavonoids, and so on, which has the characteristics of high medicinal value and small side effect. Crepis rigescens was used as folk medicines for anti-bacterial, and anti-oxidation, which also had a potential curative effect in preventing cardiovascular disease and anti-tumor. Illumina paired-end reads data were used to assemble the complete chloroplast (cp) genome. 14,425,796 raw paired-end reads and the length distribution in 124,685 bp, including a large single copy (LSC) region of 82,924 bp, a small single copy (SSC) region of 18,150 bp, and a pair of inverted repeat (IRs) regions of 25,128 bp. Besides, 10 protein-coding genes (PCGs) genes and 6 tRNAs genes possess a single intron, while clpP and ycf3 have a couple of introns. Based on the concatenated coding sequences of cp PCGs, the phylogenetic analysis showed that C. rigescens and Hypochaeris radicata (MH746729) are closely related to each other within the family Cichorioideae.

The complete chloroplast genome sequence and phylogenetic position of Lilaeopsis chinensis (L.) Kuntze (Apiaceae).

Lilaeopsis chinensis (L.) Kuntze is a traditional Chinese medicinal plant for treating diuresis, abdominal pain and eczema. In this study, its complete chloroplast genome was assembled from the whole genome Illumina sequencing data. The circular genome is 153,162 bp long, and comprises a pair of inverted repeat regions (IRs, 25,072 bp each), a large single-copy region (LSC, 84,288 bp) and a small single-copy region (SSC, 18,730 bp). It encodes a total of 113 genes (79 protein-coding, 30 tRNA and 4 rRNA genes), with 19 of them occurring in double copies. Introns were detected in 11 protein coding genes (PCG) and 6 tRNA genes. The nucleotide composition is asymmetric (30.9% A, 19.2% C, 18.4% G and 31.5% T) with an overall A + T content of 62.4%. Phylogenetic analysis challenged the traditional taxonomic framework of the family Apiaceae, and indicated that Lilaeopsis chinensis (L.) Kuntze is closely related to Hydrocotyle verticillata.

The complete chloroplast genome sequence of Rotheca myricoides (Hochst.) Steane & Mabb., a traditional medicinal plant.

Rotheca myricoides (Hochst.) Steane & Mabb. is a plant species used in traditional medicine for the management of diabetes in the lower eastern part of Kenya (Kitui, Machakos and Makueni Counties, Kenya) that is mainly inhabited by the Kamba community. The complete chloroplast genome sequence of R. myricoides was assembled from the whole genome Illumina sequencing data. The genome was 150,596 bp in length, contained an SSC region of 17,237 bp and LSC region of 83,489 bp, separated by IRs of 24,935 bp, each. The genome contained 114 unique genes, including 80 PCGs, 4 rRNA genes, and 30 tRNA genes. In addition, 18 genes contained one or two introns, including 10 PCG genes with a single intron, 2 PCG genes harboring two introns, and 6 tRNA genes harboring a single intron. Phylogenetic analysis supported R. myricoides had the closest genetic relationship with Rotheca serrata and clustered with the Rotheca family species.

Comparative proteomic analysis of latex from Euphorbia kansui laticifers at different development stages with and without UV-B treatment via iTRAQ-coupled two-dimensional liquid chromatography-MS/MS.

Euphorbia kansui Liou, an endemic species in China, is well-known in traditional Chinese medicine. All parts of E. kansui contain white latex, which is the protoplasm constituent of specialised cells known as laticifers. The latex contains many proteins with various biological functions. In the present study, isobaric tagging for relative and absolute quantitation (iTRAQ) and MS technology combined with western blot and quantitative real-time PCR analysis were used to identify latex proteins and analyse differentially accumulated proteins in laticifers at different development stages, with and without UV-B treatment according to the E. kansui transcriptome database and the NCBI Euphorbiaceae RefSeq protein database. A total of 322 latex proteins were successfully identified. Proteasome subunits, ubiquitinated proteins, vacuolar ATP synthase (V-ATPase) and lysosomal enzymes decreased, keeping the content at a higher level in laticifers in the early development stage. These results suggest that the ubiquitin-proteasome pathway and the lysosome autophagy pathway were involved in the partial degradation of laticifer cytoplasm. In addition, terpenoid biosynthesis-related proteins, 14-3-3 protein, V-ATPase and lysosomal enzymes increased under UV-B treatment, which showed that partial cytoplasmic degradation is positively correlated with secondary metabolite synthesis in the development of E. kansui laticifers. Besides, UV-B radiation can increase plant resistance by promoting laticifer development in E. kansui. This information provides a basis for further exploration of E. kansui laticifer development, and terpenoid synthesis and regulation.

iTRAQ-Based Proteomics Analysis of Autophagy-Mediated Responses against MeJA in Laticifers of Euphorbia kansui L.

Autophagy is a well-defined catabolic mechanism whereby cytoplasmic materials are engulfed into a structure termed the autophagosome. Methyl jasmonate (MeJA), a plant hormone, mediates diverse developmental process and defense responses which induce a variety of metabolites. In plants, little is known about autophagy-mediated responses against MeJA. In this study, we used high-throughput comparative proteomics to identify proteins of latex in the laticifers. The isobaric tags for relative and absolute quantification (iTRAQ) MS/MS proteomics were performed, and 298 proteins among MeJA treated groups and the control group of Euphorbia kansui were identified. It is interesting to note that 29 significant differentially expressed proteins were identified and their associations with autophagy and ROS pathway were verified for several selected proteins as follows: α-L-fucosidase, ß-galactosidase, cysteine proteinase, and Cu/Zn superoxide dismutase. Quantitative real-time PCR analysis of the selected genes confirmed the fact that MeJA might enhance the expression of some genes related to autophagy. The western blotting and immunofluorescence results of ATG8 and ATG18a which are two important proteins for the formation of autophagosomes also demonstrated that MeJA could promote autophagy at the protein level. Using the electron microscope, we observed an increase in autophagosomes after MeJA treatment. These results indicated that MeJA might promote autophagy in E. kansui laticifers; and it was speculated that MeJA mediated autophagy through two possible ways: the increase of ROS induces ATG8 accumulation and then aotophagosome formation, and MeJA promotes ATG18 accumulation and then autophagosome formation. Taken together, our results provide several novel insights for understanding the mechanism between autophagy and MeJA treatment. However, the specific mechanism remains to be further studied in the future.

MicroRNAome Profile of Euphorbia kansui in Response to Methyl Jasmonate.

miRNAs play vital regulatory roles in different plant developmental stages and in plant response to biotic and abiotic stresses. However, information is limited on the miRNA regulatory mechanism to methyl jasmonate (MeJA). In this study, we used the microRNAome profile to illustrate the relevant regulatory mechanisms of Euphorbia kansui in response to methyl jasmonate (MeJA) through Illumina RNA-Seq. As a result, we identified 875 miRNAs corresponding to 11,277 target mRNAs, among them, 168 known miRNA families representing 6019 target mRNAs sequences were obtained. 452 miRNA-mRNA pairs presented an anti-correlationship (Cor < -0.50 and p-value of correlation ≤ 0.05). The miRNA with a fold change ≥ 2 and a p (p-Value) < 0.05 in pairwise comparison were identified as significant differentially expressed miRNAs (DEMs). The DEMs in MeJA treatment of 0, 24, 36 and 48 h were compared by using Short Time Expression Miner (STEM) cluster and 4 significant gene profiles (p-value ≤ 0.02) were identified. Through the kyoto encyclopedia of genes and genomes (KEGG) pathway and gene ontology (GO) enrichment analysis on all miRNA targets, we identified 33 mRNAs in terpenoid biosynthesis, which were regulated by miRNAs under MeJA treatment, so the miRNA maybe involved in the response of E. kansui plant to exogenous MeJA and the results would provide very useful information on illustrating the regulatory mechanism of E. kansui and also provide an overall view of the miRNAs response to MeJA stress of a non-model plant.

Lysosome and proteasome pathways are distributed in laticifers of Euphorbia helioscopia L.

At present, the lysosome pathway (LP) and proteasome pathway (PP) are known as major clearance systems in eukaryotic cells. The laticifer, a secretory tissue, degrades some cytoplasm during development. In this study, we investigated the distribution of LP and PP in non-articulated laticifers of Euphorbia helioscopia L. Electron microscopy revealed that, plastids, mitochondria and some cyotsol were degraded in the late development laticifers, where there were numerous vesicles originated from dicytosomes. Accordingly, some key proteins in LP and PP were detected in E. helioscopia latex using isobaric tags for relative and absolute quantitation (iTRAQ) proteomics. Further immunohistochemistry analysis revealed that the clathrin heavy chain (CHC) belonging to LP and the ubiquitin-mediated proteasome degradation increases gradually as the laticifer develops. Immuno-electron microscopy revealed that the cysteine protease, CHC and AP-2 complex subunit beta-1 belonging to LP were mainly distributed in vesicles deriving from dicytosomes, which we called lysosome-like vesicles. Ubiquitin was widely distributed in the cytosol, and proteasome activity was significantly reduced when various concentrations of the inhibitor MG132 were added to the latex total protein. We hypothesize that LP and PP are distributed in E. helioscopia laticifers; and it was speculated that LP and PP might be involved in the degradation of organelles and some cytoplasmic matrix in E. helioscopia laticifers.

Detection of autophagy processes during the development of nonarticulated laticifers in Euphorbia kansui Liou.

MAIN CONCLUSION: Autophagy is involved in cytoplasmic degradation through directly engulfing cytosol and organelles by autophagosomes and then fusing with lysosome-like vesicles during the development of nonarticulated laticifers in Euphorbia kansui Liou. Autophagy has been reported to play an important role in a wide range of eukaryotic organisms during responses to various abiotic and biotic stresses. However, until recently, the functions of autophagy in normal plant differentiation and development were still in their infancy. Nonarticulated laticifers, a type of secretory tissue in plants, undergo the degradation of cytosol and organelles during their development. However, little evidence of autophagy in laticifer differentiation has been provided. In the present study, using anti-ATG8 antibody-Alexa Fluor 488, Lyso-Tracker Red (LTR) and monodansylcadaverine (MDC) as markers for detecting autophagosomes, as well as autophagy-related structures, we observed that the green fluorescence of ATG8a largely colocalized with the red fluorescence of LTR and purple fluorescence of MDC and the quantity of autophagosomes experienced a trend from less to more to less during laticifer development. Additionally, we described the autophagy process during the development of nonarticulated laticifers in Euphorbia kansui Liou at the ultrastructural level in detail. In addition, further immunogold TEM studies also verified the presence of autophagosomes, autolysosomes and lysosome-like structures in laticifers. Taken together, these results suggest that autophagy contributes to the development of the nonarticulated laticifers in E. kansui Liou and that autophagosomes fuse with lysosome-like structures for degradation. These results will lay an important foundation for further studies on laticifer regulation.

UPLC/Q-TOF-MS profiling of phenolics from Canarium pimela leaves and its vasorelaxant and antioxidant activities

ABSTRACT Canarium pimela K.D. Koenig, Burseraceae, have a long history of use in the Chinese traditional medicine treatment of various ailments including hypertension, and our research team has reported the anti-hypertensive activity and delineated the mechanism involved in the action. The following research aims to evaluate the vasorelaxant and antioxidant activities of ethanol extract from C. pimela leaves and to analyze its chemical composition by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) that may correlate with their pharmacological activities. The results showed that pre-incubation of aortic rings with the extract (0.3, 1, 3, 10, 30 and 100 mg/l) significantly inhibited the contractile response of the rings to norepinephrine-induced contraction (p < 0.01or p < 0.05). Crude ethanol extract and refined ethanol extract showed a highest inhibitory effect against 2,2dipheyl-2-picrylhydrazyl hydrate scavenging activity (IC50 of crude ethanol extract = 15.42 ± 0.14 µg/ml and IC50 of refined ethanol extract = 5.72 ± 0.31 µg/ml) and 2,2′-azinobis (3-ethyl-benzothiazoline-6-sulphonic acid ammonium salt) (ABTS (IC50 of crude ethanol extract = 3.24 ± 0.18 µg/ml and IC50 of refined ethanol extract = 1.88 ± 0.07 µg/ml) scavenging activity, which was considerably higher than that reported for butylated hydroxytoluene and lower of that measured for ascorbic acid. Moreover, its chemical composition was analyzed by UPLC/Q-TOF-MS. Sixteen compounds including nine flavonoids, four tannins, two phenolic acids and one dianthrone were identified for the first time as constituents of this species. And of this, six major phenolic components were simultaneous quantitative analysis by HPLC-UV, chlorogenic acid is the major compounds in C. pimela leaves. These results indicate that the phenolic-rich extract of C. pimela leaves is a promising natural pharmaceutical for combating hypertension and oxidative stress.