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Identification of genes related to agarwood formation: transcriptome analysis of healthy and wounded tissues of Aquilaria sinensis
Yanhong Xu1,
Zheng Zhang1,2,
Mengxi Wang3,
Jianhe Wei1,2,
Hongjiang Chen2,
Zhihui Gao1,
Chun Sui1,
Hongmei Luo1,
Xingli Zhang1,
Yun Yang2,
Hui Meng2 &
Wenlan Li3
BMC Genomics volume 14, Article number: 227 (2013) Cite this article
Agarwood is an expensive resinous heartwood derived from Aquilaria plants that is widely used in traditional medicines, incense and perfume. Only wounded trees can produce agarwood, and the huge demand for the agarwood products has led all Aquilaria spp. being endangered and listed in the Appendix II of the CITES (http://www.cites.org). The major components of agarwood are sesquiterpenes and phenylethyl chromones. Owing to a lack of genomic information, the molecular basis of wound-induced sesquiterpenes biosynthesis and agarwood formation remains unknown.
To identify the primary genes that maybe related to agarwood formation, we sequenced 2 cDNA libraries generated from healthy and wounded A. sinensis (Lour.) Gilg. A total of 89,137 unigenes with an average length of 678.65 bp were obtained, and they were annotated in detail at bioinformatics levels. Of those associated with agarwood formation, 30 putatively encoded enzymes in the sesquiterpene biosynthesis pathway, and a handful of transcription factors and protein kinases were related to wound signal transduction. Three full-length cDNAs of sesquiterpene synthases (ASS1-3) were cloned and expressed in Escherichia coli, and enzyme assays revealed that they are active enzymes, with the major products being δ-guaiene. A methyl jasmonate (MJ) induction experiment revealed that the expression of ASS was significantly induced by MJ, and the production of sesquiterpenes was elevated accordingly. The expression of some transcription factors and protein kinases, especially MYB4, WRKY4, MPKK2 and MAPK2, was also induced by MJ and coordinated with ASS expression, suggesting they maybe positive regulators of ASS.
This study provides extensive transcriptome information for Aquilaria spp. and valuable clues for elucidating the mechanism of wound-induced agarwood sesquiterpenes biosynthesis and their regulation.
Agarwood is widely used in traditional medicines as a digestive, sedative, and antiemetic drug and is also popular as incense and perfume in the Middle East, South Asia, Japan, and China. Additionally, the agarwood sculpturing for interior decoration is another important aspect of its value which generates a lot of income in Asia. In the international market, high-quality agarwood is more costly than gold. Agarwood is a dark, resinous, non-timber wood that forms in the stem, branch, or root of Aquilaria and Gyrinops trees after they are wounded and infected by a fungus (under natural conditions, the wounds can be caused by wind, lightning strikes, the gnawing of ants or insects, or microorganism invasion), but these natural processes always develop very slowly over decades. Owing to the economic value and great demand of agarwood, natural Aquilaria forests have been destroyed in almost all countries in which agarwood is commercially exploited. For the protection of wild Aquilaria resources and their sustainable use, all Aquilaria spp. have been listed in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (http://www.cites.org [accessed 7 August 2012]), and Aquilaria cultivation has been drawn much attention in countries such as China, India, Vietnam, Indonesia, Malaysia, and Thailand. However, the current methods used by farmers, including partial trunk pruning, burn-chisel-drill, and fungi inoculation, all require considerable time to produce agarwood and result in a product with very low yield and quality.
Aquilaria sinensis (Lour.) Gilg is one of the most important plant resources for producing agarwood in China as well as the only certified source for agarwood listed in China Pharmacopoeia[1]. More than 20 million planted A. sinensis trees are estimated to be scattered throughout Hainan, Guangdong, and Yunnan Provinces, and more than one-fourth of them are older than 5 years and have become available for agarwood induction. Uncovering the mechanism of agarwood formation in wounded trees is crucial to establishing an efficient induction method for agarwood.
Studies have shown that sesquiterpenes and phenylethyl chromone derivatives are the main compounds in agarwood [2–7]. Thus, understanding the biosynthesis and regulation of sesquiterpenes and chromone in Aquilaria spp. is critically important in determining the mechanism of agarwood formation. However, to date, the biosynthesis pathway of chromone derivatives remains almost unknown. The terpenoid metabolism pathway is, by comparison, very clear. The biosynthesis of sesquiterpenes can reportedly occur via the mevalonic acid (MVA) [8, 9] and 1-deoxy-d-xylulose-5-phosphate (DXP) [10] pathways (Additional file 1: Figure S1), in which sesquiterpene synthases are enzymes used in the final step to form sesquiterpenes. Currently, many studies on the functional identification and regulation of these enzymes have been reported. In Gossypium arboreum, GaWRKY1 positively regulates the expression of (+)-δ-cadinene synthase, which catalyzes the branch point leading to biosynthesis of sesquiterpene gossypol [11]. AaWRKY1 regulates the expression of amorpha-4,11-diene synthase (ADS), which is the key artemisinin biosynthesis enzyme in Artemisia annua. Overexpression of AaWRKY1 in tobacco or in A. annua can significantly activate ADS promoter activity, and artemisinin production increases accordingly [12]. The latest report on Arabidopsis has shown that transcription factor (TF) MYC2 interacts with DELLA proteins, positively regulating the expression of sesquiterpene synthases TPS21 and TPS11 in the jasmonic acid (JA) and gibberellic acid (GA) signaling pathways [13]. However, studies on agarwood sesquiterpenes in Aquilaria spp. are still extremely limited, and with the exception of five δ-guaiene synthase genes (AcC1, AcC2, AcC3, AcC4, and AcL154), which were cloned from cell cultures of A. crassna[5], most of the genes remain to be cloned. Kumeta & Ito [5] have also found that methyl jasmonate (MJ) treatment increases the expression of these genes and induces the production of sesquiterpene δ-guaiene. Their report was the first and remains the only molecular report of sesquiterpene biosynthesis and regulation in Aquilaria spp..
Agarwood is produced after an Aquilaria tree is wounded [14–17]. We have observed that in the early period of agarwood formation, the resin in agarwood exists in 2 forms: tylose and gel (our unpublished data), and the sesquiterpenes in agarwood have antimicrobial and anti-disease activity [6]. Based on previous studies, we have hypothesized that agarwood is the product of a plant defense response [18]: when an Aquilaria tree is wounded, damage signals are induced and transmitted, activating the defense response, after which defensive substances such as sesquiterpenes and phenylethyl chromone derivatives are produced. When these products combine with the wood tissue to avoid damage expansion, agarwood forms. The key to testing this hypothesis is to reveal the mechanism of wound-induced biosynthesis of agarwood sesquiterpenes, but this goal is hindered by the very limited amount of genomic information available for Aquilaria. The present study made use of 454 sequencing technology in order to (i) provide detailed insight into the transcriptomes of healthy and wounded A. sinensis, (ii) discover candidate transcripts with significant homology to important enzymes in the sesquiterpene biosynthesis pathway and verify their function, and (iii) identify candidate regulators that might be related to wound signal transduction and regulation of agarwood formation.
Transcriptome sequence assembly and annotation
Two cDNA libraries from the stems of the healthy and wounded A. sinensis (designated H and W) were constructed using Switching Mechanism At 5' end of RNA Transcript (SMART) technology. The 2 libraries were sequenced in one run using 454 GS-FLX pyrosequencing technology. A total of 562,060 (H) and 565,008 (W) raw reads were generated (Table 1). After the removal of sequences shorter than 100 bp and empty vectors, 1,073,469 high-quality reads [NCBI GEO, accession no. GSE42246], including 530,438 (94.37% of the raw reads) from the H library and 543,031 (96.11% of the raw reads) from the W library, were obtained. After de novo assembly of these reads, 67,972 unigenes from the H library and 74,139 unigenes from the W library were generated, respectively (Table 1). Combining the reads from the 2 libraries, we obtained 89,137 unigenes with an average length of 678.65 bp. More than 70% of these unigenes were between 200 bp and 600 bp long (Figure 1), and almost 17% contained more than 10 reads (Additional file 2: Table S1).
Length distribution for Aquilaria sinensis unigenes. Over 70% of these unigenes are between 200 bp and 600 bp long.
Table 1 Characteristics of the reads from the healthy and the wounded A . sinensis
The BLAST annotation of the 89,137 unigenes indicated that 22,051 (24.7%) shared homology with the sequences in the NCBI nucleotide database, and 38,159 (42.8%) had matches to known protein sequences in the NCBI non-redundant database. To obtain more information, we also carried out the BLASTX search against the SWISS-PROT protein database, and 17.1% of the unigenes were thus annotated (Additional file 3: Table S2), indicating that the information about the genomes and transcriptomes of Aquilaria spp. was very limited. Thus, our 454 database is important, and the unigene sequences obtained in this study may represent most of the novel genes of Aquilaria spp..
Based on an InterPro analysis, 48,530 (54%) unigenes were annotated with at least one GO term, and the distribution of gene functions based on the cellular component, molecular function, and biological process categories was very similar in the 2 libraries (Additional file 4: Figure S2). KEGG annotation showed that, in the metabolism process, although the W library had a total number of unigenes smaller than that of the H library, it had a slightly higher number of unigenes related to carbohydrate metabolism and metabolism of terpenoids and polyketides (Figure 2). This result indicated that the pruning wound in A. sinensis had activated carbohydrate, terpenoid, and polyketides metabolism, which may be related to agarwood formation.
Comparison of KEGG metabolism categories in the wounded and healthy tissue libraries.
To investigate the differences between the 2 libraries, IDEG6 [19] was used to identify differentially expressed genes (DEGs). A total of 47,169 DEGs were obtained, including 14,988 and 21,165 expressed uniquely in the H and W libraries, respectively; 11,016 were expressed in both libraries but at different levels (Figure 3). KEGG classification showed that the number of "up" unigenes was far greater than the number of "down" unigenes (the relative abundance of the W library vs. the H library: >1 marked "up", <1 marked "down"; Figure 4): for metabolism and genetic information processing, 814 (82.5%) and 652 (83.8%) of the DEGs were "up", respectively. The percentage and distribution of DEGs annotated with GO terms are shown in Additional file 5: Figure S3.
Comparison of the unigenes expressed in the healthy and wounded tissue libraries. The overlapping section represents the 52,974 (59.43%) unigenes that were found in both libraries; 14,988 (16.83%) unigenes were expressed only in the healthy library, and 21,165 (23.74%) unigenes were expressed only in the wounded library.
Functional classification of differentially expressed genes based on KEGG classification. In the categories metabolism, genetic information processing, environmental information processing, and cellular processing, the number of "up" unigenes was far greater than the number of "down" unigenes (the relative abundance of wounded library vs. healthy library >1 marked "up", <1 marked "down").
Enzyme genes involved in sesquiterpenes biosynthesis
The biosynthesis of sesquiterpenes occurs via the MVA [8, 9] and DXP [10] pathways, and 4 key enzymes are commonly involved: 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the first key enzyme in the MVP pathway; DXP synthase (DXPS), the first rate-limiting enzyme in the DXP pathway; farnesyl diphosphate (FPP) synthase (FPS); and sesquiterpenes synthases. In our 454 unigene database (H + W), 30 unigenes were annotated as being related to the sesquiterpene biosynthesis pathway, and 22 of them putatively encode these enzymes, including 3 HMGRs, 12 DXPSs, 2 FPSs, and 5 sesquiterpene synthases (Table 2). Unigenes putatively encoding other enzymes in the sesquiterpene biosynthesis pathway were also found, including acetyl-coenzyme A acyltransferase (ACTC), mevalonate kinase (MK), phosphomevalonate kinase (PMK), and 2-C-methyl-d-erythritol-2, 4-cyclodiphosphate synthase (MCS) (see Table 2).
Table 2 Number of unigenes and 454 annotated reads involved in sesquiterpenes biosynthesis
FPS is a branch point enzyme in terpenoid biosynthesis [20–22]. Its product, FPP, is the common precursor of sesquiterpenes, steroids, and farnesylated proteins. We found two unigenes that putatively encode FPS. One is a full-length cDNA (named AsFPS) that has an open reading frame of 1029 nucleotides and encodes a protein of 342 amino acids. The phenetic relationship between AsFPS and the FPSs characterized in other species is depicted in Additional file 6: Figure S4. AsFPS had the highest homology with the FPS sequence from Aquilaria. microcarpa (GenBank ID: ADH95185; 97% identity, with a difference of 5 amino acids), suggesting that AsFPS belongs to the FPS family.
With the exception of one FPS, the full-length open-reading frames (ORFs) of other unigenes were unavailable. Sesquiterpene synthases are the key enzymes that catalyze FPP, leading to sesquiterpenes biosynthesis. Therefore, they were the focus of this study.
Cloning and functional identification of A. quilaria sesquiterpene synthase genes (ASSs)
Based on the degenerated primers, 3 full-length cDNAs of sesquiterpenes synthases (ASS1, ASS2, and ASS3) were cloned and deposited in the NCBI database under the accession numbers JQ712682, JQ712683, and JQ712684. They have ORFs of 1644 nucleotides, which encode almost the same protein of 547 amino acids. Their nucleotide and protein sequences have more than 92% identity with one another. The alignment of their nucleotide and deduced amino acid sequences are shown in Additional file 7: Figures S5 and Additional file 8: Figures S6, respectively. These proteins contain 2 motifs (the RRx8W motif at the N-terminus and the DDxxD motif known to be a divalent metal ion substrate-binding site) that are functionally important and highly conserved in all terpene synthase proteins. The phenetic relationship between the 3 putative proteins and other characterized sesquiterpenes synthases is shown in Additional file 9: Figure S7. ASS1-3 were grouped with ACY38194, ACY38195, ACY38196, ACY38197 [5], AEG77018, AEG77021, and AEG77019 [23], which were cloned from A. crassna.
To confirm that ASS1-3 encode active ASSs, we cloned them into a pET-28a vector and heterologously expressed them in E. coli. The fusion proteins were detected in soluble fractions (data not shown). The enzyme assays were performed using the geranyl diphosphate (C10), FPP (C15), and geranylgeranyl diphosphate (C20) substrates, and the reaction products were analyzed using GC-MS. The result showed that ASS1-3 did not accept geranyl diphosphate or geranylgeranyl diphosphate as a substrate, converting only FPP to terpene products. The 3 enzymes yielded the same compounds; the major product was identified as δ-guaiene (74.2%), and the minor products as β-elemene (16.3%) and α-guaiene (9.5%) (Figure 5).
Gas chromatography-mass spectroscopy profiles. (A) Total ion chromatogram of the products formed by sesquiterpene synthases (ASSs) with farnesyl diphosphate as a substrate. (B) Mass spectra of the sesquiterpenes and their authentic standards. Peak 1 is β-elemene, peak 2 is α-guaiene, and peak 3 is δ-guaiene.
Expression of ASSsinduced by MJ and identification of sesquiterpenes produced in MJ-treated calluses
We analyzed the expression profile of ASS using RT-PCR. Two pools of cDNAs, one derived from the wounded stems of the 3-year-old A. sinensis that comprised the 454 library and another from A. sinensis calluses treated with MJ, were used for the analysis. MJ is an essential signaling molecule that modulates plant secondary metabolism [24–26] and is capable of inducing the production of sesquiterpenes in Aquilaria spp. [5, 27, 28]. Therefore, MJ-treated calluses were also used to investigate the expression of ASS. The results demonstrated that ASS1 and ASS2 were both induced by either mechanical wounding or MJ treatment (Figure 6A, 6B). The expression of ASS1 was upregulated significantly—approximately 800 times in response to mechanical wounding (see Figure 6A) and 1000 times in response to MJ treatment (see Figure 6B).
Expression of 4 synthase genes in response to mechanical wounding and methyl jasmonate (MJ) treatment. (A) In the stem of 3-year-old A. sinensis trees, mechanical wounding increased the mRNA levels of 4 genes that encode enzymes in the sesquiterpene biosynthesis pathway. Each value is the mean ± SE of 3 independent biological determinations. CK: healthy 3-year-old control trees; 5d: 3-year-old trees pruned after 5 days. (B) In A. sinensis calli, treatment with 100 μM MJ increased the mRNA levels of 4 genes that encode enzymes in the sesquiterpene biosynthesis pathway. A. sinensis calli were transferred to MJ-containing medium and cultivated for 5 days before being sampled for analysis. Each value is the mean ± SE of 3 independent biological determinations, and the data shown are relative ratios. The genes were named according to their functional annotation. CK: healthy calli controls; MJ: calli treated with MJ for 5 days.
Furthermore, we tested the expression of other enzyme genes in sesquiterpene biosynthesis, including HMGR, DXPS, and FPS. They were also induced by mechanical wounding and MJ, but the upregulation rate was significantly weaker than that associated with ASS1 and ASS2, fully demonstrating that ASS1 is a typical stress-inducible gene. This result is consistent with those of solid-phase micro-extraction GC-MS analysis, showing that the production of sesquiterpenes increases in response to MJ treatment. In the untreated healthy callus samples, only 2 sesquiterpenes (δ-guaiene and α-guaiene) were detected, with total peak area of 11.47. In the MJ-treated callus samples, 4 sesquiterpenes were detected, with total peak area of 850.6—approximately 74 times higher (Table 3). Among the 4 sesquiterpenes, the content of δ-guaiene and α-guaiene was more than 88%, an increase of more than 67-fold relative to the healthy control. The results demonstrated that inducible expression of ASS is responsible for the formation of agarwood sesquiterpenes in A. sinensis.
Table 3 Kinds and relative contents of sesquiterpenes from the untreated healthy calli and the MJ-treated calli
Candidate regulators response to wound signals
TFs are critical regulators of gene expression and environmental stress responses [26, 29]. In our 454 unigene database (H + W), a total of 4,786 unigenes represented homologs of various TF families (Additional file 10: Table S3): 86 were annotated as belonging to the APETLA2 (AP2)/ethylene-responsive-element-binding family [30, 31], 66 to the bZIP family [32], 108 to the MYB family [33–35], 150 to the MYB-related family, 169 to the bHLH family [35], and 99 to the WRKY family [36, 37]. All of these TFs are reportedly stress-related [36–39], and some homologous genes have been identified as positive or negative regulators in the biosynthesis of secondary metabolites in other plants [11, 40–42].
In defensive response and wound signal transduction, calcium signaling and mitogen-activated protein kinase (MAPK) cascades play important roles [25, 43, 44]. In our study, KEGG classification annotated 25 unigenes as being related to calcium signaling pathways and 41 to MAPK signaling pathways (Additional file 11: Table S4).
RT-PCR analysis of candidate regulators response to wound signals
To confirm which of the candidate regulators mentioned above are related to wound signals, we performed RT-PCR analysis to test the effects of MJ on the expression of these genes. Overall, 30 expressed regulators were selected (Additional file 12: Table S5), including TFs MYB and WRKY, protein kinases CDPK and MAPK, and some regulators related to signal molecules MJ, ethylene, and hydrogen peroxide. As shown in Figure 7, the expression of most of these genes was elevated in response to MJ treatment, and only 4 TFs, HD-ZIP, WRKY23, WRKY60, and WRKY32, were downregulated by MJ. Among the upregulated genes, 17 were increased more than 10-fold. They were TFs WRKY4 and MYB4, protein kinases CDPK1, CDPK2, CDPK5, CDPK6, MPKK2, MAPK2, MPK3, MAPK5, and JA-, ethylene-, or hydrogen peroxide-related regulators JAip, RBF, EIN3, ERF115, noxB, AOSC, ACO3. The expression of 5 annotated unigenes, MPKK2, MAPK2[45], MYB4[46], WRKY4, and noxB, the superoxide-generating NADPH oxidase [47, 48], was coordinate with ASS1, showing elevation of 126.9-, 63.9-, 80.9-, 23.7-, and 95.2-fold, respectively. The expression profiles of these wound-signaling-related genes are essentially consistent with the idea that the expression of ASS1 is induced by wounding but regulated through a complex underlying mechanism.
Altered expression of a set of regulatory genes in response to MJ treatment. Treatment with 100 μM MJ altered the mRNA levels of a set of regulatory genes that may be involved in wound signal transduction. Each value is the mean ± SE of 3 independent biological determinations, and the data shown are relative ratios. The genes were named according to their functional annotation. CK: healthy calli controls; MJ: calli treated with MJ for 5 days.
Aquilaria sinensistranscriptome sequencing
Although Aquilaria is the most important plant source of agarwood and all Aquilaria spp. are listed in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora [49], little progress to date has been made in deciphering the mechanism of agarwood formation and genomic and genetic information is lacking. The NCBI EST database contained only a few hundred Aquilaria sequences before this study. The 454 pyrosequencing reported herein represents the first concrete effort to generate cDNA resources for Aquilaria spp. We obtained 89,137 unigenes from the H and W A. sinensis cDNA libraries (Table 1) and annotated them in detail at bioinformatics levels (see Additional file 3: Table S2, Additional file 4: Figure S2 and Additional file 5: Figure S3). We estimated the genome size of A. sinensis preliminarily at approximately 2 G using flow cytometry. In the absence of a haploid, genome sequencing is difficult to carry out with current sequencing technology. In the absence of complete genome sequences, transcriptome mining provides the sole option for the subsequent annotation of sequences. The data reported herein may be useful for further research on the functional genomics of Aquilaria spp.. Importantly, by comparing the unigenes in the W and H libraries, we obtained a large number of DEGs (see Figure 3). These data suggested that the transcriptome of A. sinensis changed significantly in response to mechanical damage and also provided resources for screening genes related to wound signaling and metabolism. KEGG annotations of these DEGs showed that most of the unigenes were related to metabolism processes, especially carbohydrate metabolism, and had higher abundance in the W library than in the H library (see Figure 4). The number of carbohydrate metabolism-related unigenes was slightly greater in the W library, indicating that starch and sugar metabolism in A. sinensis may be stimulated by wounding (see Figure 2).
The synthesis of sesquiterpenes starts with acetyl-coenzyme A (the MVA pathway) [8] or glyceraldehyde-3-phosphate and pyruvate (the DXP pathway) [10] from glycolysis. Therefore, agarwood formation may have some association with starch and sugar metabolism and is essentially consistent with our electron microscopy findings that a high number of starch granules exist in the healthy wood of A. sinensis, but when the wood is wounded, resin in the agarwood accumulates while the starch granules degrade (our unpublished data).
ASSsare inducible enzymes that may be responsible for the formation of agarwood sesquiterpenes
Considering that sesquiterpenes are the most important components of agarwood [2, 6, 7], we tried to identify the genes that encode the enzymes involved in sesquiterpene biosynthesis, and 30 such unigenes were found in our database, including 4 categories of critical genes (HMGR, DXPS, FPS, and ASS) and other genes encoding synthases in sesquiterpene metabolism in Aquilaria spp. (Table 2). These data will be valuable for further studies of the terpene biosynthesis pathway. Using a homology-based PCR approach, we obtained 3 full-length cDNAs of ASSs (ASS1-3) with very similar amino acid sequences. Phenetic analysis showed that the evolutional distances among the 3 ASSs were quite small compared to those of sesquiterpene synthases from other plant species, except A. crassna (see Additional file 9: Figure S7). Heterologous expression in E. coli and enzyme assays showed that ASS1-3 had ASS activity, and the major product was revealed to be δ-guaiene (see Figure 5). Expressions analysis with real-time PCR showed that ASS1 was barely detectable in either the healthy tissues or the calluses of A. sinensis, whereas in the wounded tissues or MJ-treated calluses, ASS1 expression was increased almost 1000-fold (see Figure 6). Furthermore, GC-MS analysis showed that the major product was δ-guaiene, essentially consistent with its enzyme reaction, and the sesquiterpene content was increased 74-fold (Table 3), clearly demonstrating that ASS1 expression was wound-induced and is responsible for the formation of agarwood sesquiterpenes in A. sinensis.
Previous reports have shown that MJ can induced 3 sesquiterpenes (α-guaiene, α-humulene, and δ-guaiene) in cultured cells of A. crassna[5, 27, 28] and be produced by the enzymatic reaction of cloned enzymes. Of the 5 clones (AcC1, AcC2, AcC3, AcC4, and AcL154), AcC1 and AcL154 have no TPS activity that might indicate pseudogenes [5]. The 3 clones (ASS1-3) in this study have high homology with AcC4 and AcC3 (see Additional file 9: Figure S7) and catalyzed the same major product. Thus, the present experiments allow us to identify ASS1-3 as active enzymes of A. sinensis, and their inducible expression is responsible for the formation of agarwood sesquiterpenes.
Wounding induces ASSsexpression via a complex signaling pathway
Research has shown that only after wounding (including mechanical wounding, chemical induction, and fungal infection, among others) can agarwood be induced in the healthy wood of Aquilaria spp.[6, 14–17]. It is, therefore, reasonable to suppose that the early period of agarwood formation is a process of wound signaling transduction before the strong expression of ASSs. Previous studies in other plant species have shown that TFs play important roles in signal transduction and gene expression. Some TFs, such as WRKY, MYB, MYC, bZIP, ERF, and EIN3 are involved in plant defense and stress responses [34, 36, 39]. Our 454 unigene database contained 4,786 putative TFs. Some homologous genes of these TFs, such as AP2, WRKY, and MYC, have been identified as positive regulators of sesquiterpene synthases in other plant species [11–13, 42]. In Catharanthus roseus, the TF ORCA3 regulates the biosynthesis of terpenoid indole alkaloids and plays an important role in JA response [42]; in cotton, the TF GaWRKY1 positively regulates the expression of the sesquiterpene synthase (+)-δ-cadinene synthase gene and the synthesis of gossypol sesquiterpene [11]. Similarly, in A. annua, AaWRKY1 and 2 JA-responsive AP2/ethylene response factor (ERF) TFs, ERF1 and ERF2, are involved in regulating the expression of the sesquiterpene synthase ADS and the production of artemisinin [12, 49]; in Arabidopsis, MYC2 interacts with DELLA proteins and positively regulates the expression of TPS21 and TPS11 [13].
Reversible protein phosphorylation has been proven to be an important regulatory mechanism in the wound signaling pathway, and studies on Arabidopsis, tomato, and tobacco have indicated that the MAPK cascade is a widespread pathway [44]. In addition, calcium and calcium-binding proteins may play some roles in the regulation of early wound responses by elevating intracellular calcium levels and changing the phosphorylation patterns of proteins [50]. In this study, 41 unigenes were annotated as related to the MAPK pathway and 25 to calcium signaling, which may play roles in the wound response of agarwood formation.
In the present experiment, 30 DEGs related to wound signal were selected to investigate whether they are co-expression with ASS1 in response to MJ. The results showed that 26 were upregulated and 4 were downregulated. Of these, 5 unigenes, including the TFs MYB4 and WRKY4, the protein kinases MAPK2 and MPKK2, and the NADPH oxidase noxB, were significantly upregulated by MJ. The downregulated genes were WRKY23, WRKY32, WRKY60, and HD-ZIP (see Figure 7). The ASS1 promoter has been cloned and scanned with PLACE (http://www.dna.affrc.go.jp/htdocs/PLACE/signalscan.html). It contains some cis-acting elements that could be combined specifically by certain TFs, including WRKY, MYB, and MYC (data to be published elsewhere). Therefore, we speculate that TFs MYB4 and WRKY4 may positively regulate the expression of sesquiterpene synthase, and the downregulated genes may negatively regulate its expression. The generation of hydrogen peroxide has been confirmed to occur both locally and systematically, and its main source is the plasma-membrane-bound NADPH oxidase. In the present study, NADPH oxidase noxB was significantly upregulated by MJ, which is consistent with the theory that MJ induces the production of hydrogen peroxide [51–54]. MAPK signal transduction pathways are widespread mechanisms in eukaryotic cells that couple environmental responses and transcriptional regulation [55, 56]. Two coupled components of the MAPK cascade, MAPK2 and MAPKK2, were significantly upregulated by MJ, demonstrating they may be involved in MJ-mediated signaling. Calcium is a prevalent messenger molecule in higher plant cells that can mediate plant responses to external signals and regulate a variety of physiological processes. In our experiments, 4 CDPKs (CDPK1, CDPK2, CDPK5, CDPK6) were upregulated more than 10-fold (see Figure 7).
Based on these results, we proposed a model to explain the mechanism by which ASS1 is modulated at the transcriptional level (Figure 8). In this model, the MAPK cascade is activated by wound signals and phosphorylates downstream TFs such as MYB or WRKY. JA signaling may activate the TFs downstream via the hydrogen peroxide or JA-JAZ-TF pathways [57]. Activated TFs bind to the cis-acting element in the promoter of ASS1 and initiate ASS1 transcription. ASS2 is also inducible and may be influenced by the same regulatory mechanisms as those affecting ASS1. The results reported herein enhance understanding of wound-induced sesquiterpene synthases expression and agarwood formation. Future studies will focus on verifying this proposed model based on the promoter of ASS1 that we have cloned. We will identify which TFs interact with the ASS1 promoter, confirm whether the MAPK cascade is involved in the wound signaling pathway, and determine the roles of them in ASS1 expression. Clarifying the wound-induced sesquiterpene biosynthesis pathway will help to develop strategies for controlling agarwood production and for the long-term protection of Aquilaria spp..
Proposed mechanism of wound-induced sesquiterpene biosynthesis and regulation. Wound signals stimulate the mitogen-activated protein kinase (MAPK) cascade and phosphorylate transcription factors downstream. Jasmonic acid (JA) signals occur via the hydrogen peroxide pathway or directly activate the transcription factors downstream. The activated transcription factor binds to the cis-acting element in the promoter of ASS1 and starts the transcription of sesquiterpene synthase. Dashed line indicates a possible pathway; '?' indicates an unknown factor. MYBRS, MYB recognized sequence; MYCRS, MYC recognized sequence.
A comprehensive transcriptome analysis was conducted for healthy and wounded stems of A. sinensis in this study. A total of 89,137 unigenes with an average length of 678.65 bp were obtained, and they were annotated in detail at the bioinformatics levels. These data should be useful for further research on the functional genomics of A. sinensis and other Aquilaria spp.. Transcripts related to sesquiterpene metabolism and regulation were highlighted. Three full-length cDNAs of sesquiterpene synthase (ASS1-3) were cloned and identified. Through MJ-inducible expression pattern experiments and GC-MS analysis, we found that their expression was significantly induced by MJ, and sesquiterpenes content was elevated accordingly. The expression of a number of important regulatory genes was detected using RT-PCR and indicated that some of them may participate in one or more processes of wound signal transduction, transcriptional regulation of ASS, and biosynthesis of agarwood sesquiterpenes. Based on the analysis of ASS1 promoter, we confirmed preliminarily that TFs MYB4 and WRKY4 may be key positive regulators of ASS1 expression. These results provide a better understanding of sesquiterpene biosynthesis and regulation in Aquilaria spp., and provide valuable clues and a basis for elucidating the molecular mechanism of agarwood formation.
Plant materials for cDNA library construction
Three-year-old A. sinensis trees were planted in Hainan Province of China. The stems were pruned with a knife, and samples were taken from 6 cm below the pruned site at various times after pruning: 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 3 d, and 5 d. All of the stems from the wounded plants were mixed in equal proportion to generate material for the wounded library, and the stems cut from healthy trees were used to generate material for the healthy library.
Plant materials for real-time PCR (RT-PCR) and gas chromatography–mass spectrometry (GC-MS) analysis
Calluses originating from the stems of the healthy A. sinensis plants were used to investigate the effect of MJ on candidate genes expression and sesquiterpenes production. Calluses were transferred to 100 μM MJ-containing medium and allowed to grow for 5 days before being sampled for analysis.
Library construction and 454 sequencing
Total RNA was isolated from the wounded and healthy materials in the libraries using a TRIzol kit (Invitrogen, USA) and purified to enrich messenger RNA (mRNA) and exclude transfer RNA (tRNA) and ribosomal RNA (rRNA) using an mRNA purification kit (Promega, USA) according to manufacturer instructions. The mRNA was reverse-transcribed with PowerScript II (Takara, Japan) using SMART IV oligonucleotide PCR primers (5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3′) and a CDS III/3′ PCR primer (5′-ATTCTAGAGGCCGAGCGGCCGACATG -d[T]30 N-1 N-3′). Long-distance PCR for double-stranded cDNA amplification was performed with LA Taq enzyme (Takara, Japan) for 25 cycles (95°C for 30 s, 68°C for 8 min) according to the SMART cDNA Library Construction Kit user manual. Finally, the double-stranded cDNA was purified using a DNA purification kit (Qiagen, Germany) to generate high-quality cDNA. Approximately 10 μg cDNA was sheared and used for 454 sequencing. The cDNA sample was end-repaired and ligated to adapters. Streptavidin bead enrichment, DNA denaturation, and emulsion PCR were performed according to a procedure described by Margulies [58]. Each portion of the preparations from the healthy and wounded libraries were loaded on a full plate and sequenced in one run.
454 data processing and assembly
A Perl script was used to remove vector sequences and Poly(A)/(T) tails from the original sequences. Reads shorter than 100 bp were deemed low quality reads and removed before assembly. The remaining high-quality reads were assembled using MIRA [59] to construct unique consensus unigenes, with the parameter --job = de novo, est, normal, 454 -SK: mnr = yes -SK: rt = 2454_SETTINGS -LR: mxti = no, and then to remove the redundancy of unigenes, UIcluster [60] (version 2.0) was used to cluster the unigenes, with the parameter --tryRevC -R 50 –M 40 –E 2, finally the clusters of unigenes were assembled by Cap3 [61], with parameter –o 100 –p 95.
Functional annotation and classification
NT and NR
The unigenes were searched against the NCBI's (http://www.ncbi.nlm.nih.gov) Non-redundant nucleotide database (NT) using the BLASTN program and against the Non-redundant protein database (NR) using the BLASTX program. For both searches, an E value ≤ 1e-5 was used.
SWISS-PROT
The unigenes were searched against the SWISS-PROT database (http://www.expasy.ch/sprot) using the BLASTX program with an E value ≤ 1e-10.
KEGG and KEGG Orthology
Unigenes were compared with the Kyoto Encyclopedia of Genes and Genomes database (KEGG, release 50; http://www.genome.jp/kegg/) [62] using the BLASTX program with an E value ≤ 1e-10. A Perl script was used to retrieve KEGG Orthology (KO) information from the BLAST results and then to establish pathway associations between the unigenes and KEGG.
Interpro and Gene Ontology
InterProScan [63] release 16.0 was used to annotate the InterPro domains of the unigenes, and then the functional assignments were mapped onto the Gene Ontology (GO) [64]. The WEGO tool [65] was used to assign GO classifications and draw GO trees.
Differentially expressed unigenes detection
The IDEG6 web tool [19] was used to identify unigenes that showed statistically significant differences in relative abundance (as reflected by the total count of individual sequence reads) between the two libraries. Comparisons between the wounded and healthy samples were performed using the general Chi2' method, which has been proven to be the most efficient test. The unigenes with P ≤ 0.01 were deemed to be significantly different between the two samples. This is similar to the credibility intervals approach that was employed earlier for the analysis of SAGE data [66].
Transcription Factors annotation
Assembled transcripts were searched against all the transcription factor protein sequences of Plant transcription factor database [67] (PlnTFDB, version 3.0) using BLASTX with an E-value cut-off of ≤ 1e − 10, and positives identity ≥50%.
Phenetic analysis
The translated amino acid sequences and the putative amino acid sequences derived from the NCBI database search were aligned using the CLASTAL W program. Evolutionary distances were calculated using the Poisson correction method, and a neighbor-joining (NJ) tree was constructed with MEGA4 [68]. Bootstrap values were obtained after 1,000 replications.
Quantitative real-time PCR (RT-PCR) analysis
To investigate the expression of the mRNA of candidate genes, RT-PCR was performed using the Bio-Rad IQ-5 Real-Time System essentially according to the manufacturer's instructions. Total RNA was isolated from three-year-old A. sinensis stems and calluses using a Total RNA Purification Kit (LC Science, USA) with an on-column DNA digestion according to the manufacturer's instructions. The RNA samples were then reverse transcribed using the Superscript II RT kit (Invitrogen, USA). The amplification of the AcHistone gene [5] was used as an internal control. The efficiency of annealing of the oligonucleotide PCR primer sequences (listed in Additional file 13: Table S6) was evaluated using the Primer 5.0 program. The cDNA was amplified in a 10 μL volume using SYBR Premix Ex Taq (TaKaRa, Japan) and a DNA Engine Opticon 2 thermal cycler with the following program: one cycle of 95°C for 10 s and 40 cycles of 94°C for 10 s and 60°C for 20 s. The amplification of the target genes was monitored every cycle by SYBR-green fluorescence. The Ct (threshold cycle), defined as the PCR cycle at which a statistically significant increase of reporter fluorescence was first detected, was used as a measure for the starting copy numbers of the target gene. Relative quantization of the target gene expression level was performed using the comparative Ct method. Three technical replicates were performed for each experiment. For all the RT-PCR analyses, the assays were repeated three times along with at least three independent repetitions of the biological experiments, and the means of the three biological experiments were calculated to estimate gene expression.
GC-MS analysis and identification of sesquiterpene components in A. sinensiscalluses
GC-MS analysis was performed with a Varian 450 GC (USA) equipped with a VF-5MS capillary column (30 m × 0.25 mm internal diameter; film thickness, 0.25 μm), and a Varian 300 mass spectrometer with an ion-trap detector in full-scan mode under election impact ionization (70 eV). The carrier gas was helium, and the flow rate was 1 mL/min. The injections were performed in splitless mode at 250°C. The healthy and MJ-treated calluses were powdered and took the same weight (0.5 g) to place into the 15 mL sample bottle, and balanced for 30 min in 60°C water. The fused silica fiber of the Solid Phase Micro Extraction (SPME) was introduced into the headspace above the sample, adsorbing the volatile components for 30 min. The adsorbed components were followed by a thermal desorption process by introducing the SPME fiber into the injection port of a gas chromatography. The program was immediately started, and the fiber was removed after 10 min. The condition for GC-MS and identification of sesquiterpenes compounds were performed according to previously described procedures [6]. The two fibers were injected separately and ran in the same program, and the relative components were obtained by peak areas normalization without applying correction factors [6]. We didn't use the internal standard, for it is difficult to choose one suiting for both of the samples. Peaks areas can reflex the content of concerning compound in the two different samples in some extent, as the weight of the two samples and the analytical methods were identical.
Heterologous expression of ASS genes in Escherichia coliand enzyme assays
The full-length sequence of ASS1 was cloned into the XhoI (5′ end)/BamHI (3′ end) sites of a pET-28a vector (Novagen) and expressed in E. coli as a His-tag fusion protein using the following primers: forward primer, 5′-CCGCTCGAGATGTCTTCGGCAAAACTAGGT-3′ and reverse primer, 5′-CGGGATCCGATTTCAATAGCATGACGCAAC-3′ (XhoI and BamHI sites are indicated with underlining). To ensure that no errors were introduced by the PCR, the construct was checked with sequencing. Fusion proteins in the E. coli BL21 (DE3) cells were induced through treatment with 1 mM isopropylthio-β-galactoside (IPTG) for 12 h at 30°C and 200 rpm. The protein purification was performed using a Profinia Protein Purification System (Bio-Rad, USA). Bacterial lysates and purified enzyme samples were examined on SDS-PAGE gels to visualize expected proteins. Protein concentrations were determined with the Bradford assay using bovine serum albumin as the standard. The enzyme reaction was performed according to the method described by Kumeta & Ito (2010), with minor modification. The reaction was performed in a 4-mL vial with a solid-top polypropylene cap using 50 mL of crude protein extract in a final volume of 200 mL containing Tris–HCl buffer (25 mM, pH 7.0) supplemented with 10% glycerol, 100 mM MgSO4, 5 mM DTT, and 46 mM FPP. After incubation at 30°C for 4 h, a SPME fiber was inserted into the headspace of the vial to collect volatiles for 30 min and then transferred to the injection port of a GC-MS system for desorption. The GC-MS system was described above and the operating parameters were essentially according to Kumeta & Ito [5]. The identification of sesquiterpenes was based on a comparison of retention times and mass spectra with authentic standards.
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The authors gratefully acknowledge Haibo Sun and Tao Feng for their assistance with bioinformatics. This study was supported by the National Natural Science Foundation of China (No. 31100220, No. 81173481, No. 31000136 and No. 81001607), the Program for New Century Excellent Talents in University Funded by the Ministry of Education of China (No. 2008), National Key Technology R&D Program (No. 2011BAI01B07), Program of Modernization for Traditional Chinese Medicine of Hainan Provincial of China (No. 2012ZY002, 2010ZY001), and the Key Project in the Science & Technology Program of Hainan Provincial (No. ZDXM20120033,ZDZX20100006).
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, No. 151, Malianwa North Road, Haidian District, Beijing, 100193, China
Yanhong Xu
, Zheng Zhang
, Jianhe Wei
, Zhihui Gao
, Chun Sui
, Hongmei Luo
& Xingli Zhang
Hainan Branch Institute of Medicinal Plant (Hainan Provincial Key Laboratory of Resources Conservation and Development of Southern Medicine), Chinese Academy of Medical Sciences & Peking Union Medical College, Wanning, 571533, China
Zheng Zhang
, Hongjiang Chen
, Yun Yang
& Hui Meng
Research Center on Life Sciences and Environmental Sciences, Harbin University of Commerce, Harbin, 150076, China
Mengxi Wang
& Wenlan Li
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Correspondence to Jianhe Wei.
YHX participated in the design of the study, analyzed the data, performed the real-time PCR experiment and drafted the manuscript. ZZ contributed to the sample collection, participated in the design of the study and analysis of the data. MXW performed the cloning and identification of three sesquiterpene synthase genes. JHW initiated the project, helped conceive the study, and revised the manuscript. HJC participated in the GC-MS analysis. ZHG, HML, XLZ and WLL participated in the discussion of the result and helped to revise the manuscript. CS helped to analysis the result and with the phenetic analysis. YY and HM helped to the samples collection. All authors read and approved the final manuscript.
Yanhong Xu, Zheng Zhang, Mengxi Wang contributed equally to this work.
Additional file 1: Figure S1: Sesquiterpene biosynthesis pathway in plants. (DOCX 462 KB)
Additional file 2: Table S1: Distribution of the number of reads in the Aquilaria sinensis unigenes. (DOCX 13 KB)
Additional file 3: Table S2: Summary of annotation of the A. sinensis 454 assembled unigenes. (DOCX 13 KB)
Additional file 4: Figure S2: Comparison of healthy and wounded tissue libraries based on GO terms. (DOCX 307 KB)
Additional file 5: Figure S3: Functional classification of differentially expressed genes based on GO terms. (DOCX 254 KB)
Additional file 6: Figure S4: Phenetic analysis of the proteins translated from the full-length AsFPS sequence from Aquilaria sinensis and some characterized farnesyl diphosphate synthase sequences from other plant species. Bootstrap values after 1000 replications are shown on the branches. (DOCX 302 KB)
Additional file 7: Figure S5: Alignment of nucleotide sequences for 3 sesquiterpene synthase (ASS) genes. (DOCX 863 KB)
Additional file 8: Figure S6: Alignment of deduced amino acid sequences for 3 ASSs. (DOCX 449 KB)
Additional file 9: Figure S7: Phenetic analysis of 3 full-length sesquiterpene synthase sequences from A. sinensis (ASS1-3) and some characterized sesquiterpene synthase sequences from other plant species. Bootstrap values after 1000 replications are shown on the branches. (DOCX 19 MB)
Additional file 10: Table S3: Major transcription factors identified from A. sinensis. (DOCX 15 KB)
Additional file 11: Table S4: Unigenes annotated as being related to the calcium signaling and mitogen-activated protein kinase signaling pathways. (DOCX 13 KB)
Additional file 12: Table S5: Candidate regulators used for RT-PCR analysis. (XLSX 11 KB)
Additional file 13: Table S6: Primers used in this study. (DOCX 16 KB)
Xu, Y., Zhang, Z., Wang, M. et al. Identification of genes related to agarwood formation: transcriptome analysis of healthy and wounded tissues of Aquilaria sinensis. BMC Genomics 14, 227 (2013) doi:10.1186/1471-2164-14-227
Aquilaria sinensis
Wound signal transduction
Plant genomics


text:
in Asia, Europe, Featured, National Security, News, Politics, Sarawut News, Thailand, World News
Home World News Asia
PARIS — Cambodia's most prominent opposition leader, Sam Rainsy, was stopped from boarding a flight in Paris on Thursday in his attempt to return home to challenge his country's longtime autocratic leader, but he assured his followers he will go ahead with his plan.
He and fellow leaders of the banned Cambodia National Rescue Party had vowed to enter Cambodia from Thailand on Saturday to spark a popular movement to oust Prime Minister Hun Sen from power.
When Sam Rainsy tried to take a Thai Airways flight to Bangkok from Paris, where he lives in exile, Thailand's flag carrier told him "they had received from very high up the instruction to not allow me to board," he said.
Thai Prime Minister Prayuth Chan-ocha said Wednesday that Sam Rainsy would not be allowed to enter Thailand because members of the Association of Southeast Asian Nations — like Thailand and Cambodia — have a policy of not interfering in the affairs of neighboring countries.
Hun Sen had declared that Sam Rainsy and his colleagues would be blocked from entering Cambodia and had informed neighboring countries that they were unwelcome.
Malaysia also has hindered the free movement of Cambodian opposition politicians, stopping two party members from flying to Thailand and temporarily detaining Cambodia National Rescue Party Vice President Mu Sochua when she arrived late Wednesday night.
At Charles de Gaulle Airport outside Paris, an angry Sam Rainsy told reporters that he wouldn't be cowed by being kept off his flight and said he plans to return via another neighboring country.
"Never, never will I abandon. We need to continue, the days of Hun Sen are numbered. Democracy will be reinstalled in the near future. It's our conviction and our determination," he said.
Before heading to the airport, Sam Rainsy told The Associated Press that Hun Sen is going all out to block their return because he is "very afraid."
He later made an online broadcast on Facebook, where he has almost 4.8 million followers, calling on his compatriots not to be disappointed and remain strong.
"We will be seeking all the possibilities, all the options, in order to make sure that we are finally able to arrive in Cambodia to push for the regime change plan that our Cambodians are hungry to see," he said.
Many human rights activists criticized ASEAN countries for attempting to block Sam Rainsy and his colleagues.
"What we are seeing now shows that the long arm of Hun Sen's repression is reaching all over Southeast Asia. Members of ASEAN states are now collaborating with Hun Sen in making sure that there is no space for the opposition party and their network to launch any campaign to challenge Hun Sen," said Sunai Phasuk, a senior researcher based in Thailand for the group Human Rights Watch.
Malaysia released Mu Sochua and the two other activists on Thursday even though Prime Minister Mahathir Mohamad had earlier said Malaysia doesn't want to be used as a base for political activists or interfere in the affairs of other countries.
Mu Sochua said after her release that the Malaysian government demonstrated that it abides by its own laws and makes its own decisions.
"There is hope . there is no way my spirit can be impacted by such a detention," she told The Associated Press. "We have been struggling for democracy for the past 25 years, and I've also been in prison, in the prison of Mr. Hun Sen."
Nicholas Bequelin, Amnesty International's East and South East Asia regional director, said Malaysian authorities "have ultimately done the right thing – but the three should never have been detained in the first place. Other ASEAN states must follow suit and refuse to collude in Cambodia's human rights abuses."
Cambodian officials have repeatedly warned that if the opposition leaders make it back, they will be immediately arrested. Most if not all have convictions or charges pending against them in Cambodian courts, including inciting armed rebellion, despite their avowedly nonviolent intentions.
Cambodian courts are widely considered to be under the influence of the government, which employs the law to harass its opponents.
The Cambodian opposition party was dissolved by court order in late 2017, allowing Hun Sen's ruling Cambodian People's Party to sweep a 2018 general election.
Source: APNews
Tags: AsiaAsia Pacific Thailandbanned Cambodia National Rescue PartyCambodiaCambodian courtsCambodian opposition partycountry's longtime autocratic leaderElectionGlobal tradeHun SenParisPrayuth Chan-ochaprominent opposition leaderSam RainsySoutheast Asiastopped from boarding a flightThailand News
Questioning the urgency of Deputy Commander of Indonesian National Army

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Two low-pressure areas merge into a large flow of warm air from the south, a blast of cold from the north, and moisture feeding from the Gulf Stream, simultaneously. That's a perfect storm, a disastrous confluence of individually innocuous events. It's rare but when it happens, the negative consequences are magnified, like when Billy Bob Thornton hooked up with Angelina Jolie. Hard to believe such things can happen. But they do, and when they do, the results are. . . unpleasant. But what if a series of discrete events could add up to something positive? Something greater than the sum of its parts? It happened once right here in the Way Back Studios. I knew there was a series of songs I could link with similar sound effects, like rainstorms, and thunder, and crashing waves. I was tempted by the Who's Quadrophenia. But I resisted. Same with the Doors. "Riders on the Storm" would work, but it was too obvious. .
Then, in the South Side of the Sky I could see it. The Storm at Sunup. And a figure, a man named Gino Vannelli, in a white shirt, unbuttoned to reveal copious and, apparently combed, chest hair staring out from the album cover. In the background, a woman, languid in a slip with the strap slipping down her right shoulder. Staring out the window, chin in hand, elbow on knee, knee deep in ennui. Her profile seems to be saying, love me now. Fragile? Yes. Certainly, though not obvious at first. Still, the two records came together like a perfect storm to create a one of our finest batches of All Hand Mixed Vinyl. Two tracks each from two albums, re-imagined in seven parts as if originally conceived that way. And then, because it fit so nicely, we added a little Spirit at the end with the great sax-man Ernie Watts sitting in on the track "Stoney Night" from Spirit's album Farther Along. But first, "We Have Heaven."
Yes We Have Heaven
Gino Vannelli Storm at Sunup (part 1)
Yes South Side of the Sky (part 1)
Gino Vannelli Love Me Now
Spirit Stoney Night
Yes We Have Heaven (reprise)
You can call that what you will, but that's what I call transitional shenanigans. We spent the first twenty minutes of that set going back and forth between a couple of tracks each from Gino Vannelli's third album, Storm at Sunup and the fourth album from Yes, simply called "Fragile." The set opened with "We Have Heaven" which, at the end, slips into some storm sound effects and segues into "South Side of the Sky." But, unwilling as we are to leave well enough alone, we slipped into the storm sound effects at the start of Gino's "Storm at Sunup," a song that begins as a melodramatic ballad, transitions into a furious bit of jazz rock fusion, before returning to the melodrama and ending on a sustained synthesizer note that segues into "Love Me Now." During those transitions, we mixed in and out of "South Side of the Sky." What's really cool about it is that you never have to lift a needle on either album. The two records play like a handshake with the sound effects and other transitional elements presenting themselves as if by design. .
Some say it was pure dumb luck, others talk about instinct. Either way, it turns out you can mix back and forth between the movements of the four songs, playing them in exactly the order they appear on their respective records, with nothing left out. But when all was said and done, we were still a few minutes shy of a full set. So I pulled two more records from the shelf: one, an old sound effects album with storm sound effects, and the other, Spirit's under-appreciated L.P. Farther Along. We used storm effects to transition from "South Side of the Sky" into "Stoney Night" which opens with a few claps of thunder and the great sax work of Ernie Watts. Then, bringing the whole thing full circle, we flipped Fragile over and closed with the very end of side two. At the end of "Heart of the Sunrise", you hear the sound effect of a door opening followed by a reprise of "We Have Heaven." Bringing us right back where we started. And now we're where we end. Thanks for joining us. In the Way Back Studios, I'm Bill Fitzhugh and I'll have another batch of All Hand Mixed Vinyl soon enough, right here in the Deep Tracks.


75865 Olson Rd, Clatskanie, OR 97016 is a Miscellaneous (Residential) property with 2 bedrooms, 2 bathrooms, and is approximately 963 sq feet of living space. The estimated market value for 75865 Olson Rd, Clatskanie, OR 97016 is $177,000, based on recent sales of similar homes and other property assessment information. Built in 1947, this Miscellaneous (Residential) is located in Clatskanie, OR in the zip code 97016. The nearest zip codes are 97051 , 97054 , 97064. Westport, East Cathlamet and Puget Island are the nearest cities.
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Extremists attack Kenya military base, 3 Americans killed
by: ABDI GULED, TOM ODULA and CARA ANNA, Associated Press
Posted: Jan 5, 2020 / 01:08 AM CST / Updated: Jan 5, 2020 / 04:12 PM CST
In this photo taken Aug. 26, 2019 and released by the U.S. Air Force, airmen from the 475th Expeditionary Air Base Squadron conduct a flag-raising ceremony, signifying the change from tactical to enduring operations, at Camp Simba, Manda Bay, Kenya. The al-Shabab extremist group said Sunday, Jan. 5, 2020 that it has attacked the Camp Simba military base used by U.S. and Kenyan troops in coastal Kenya, while Kenya's military says the attempted pre-dawn breach was repulsed and at least four attackers were killed. (Staff Sgt. Lexie West/U.S. Air Force via AP)
NAIROBI, Kenya (AP) — Al-Shabab extremists overran a key military base used by U.S. counterterror forces in Kenya before dawn Sunday, killing three American Department of Defense personnel and destroying several U.S. aircraft and vehicles before they were repelled, U.S. and Kenyan authorities said.
The attack on the Manda Bay Airfield was the al-Qaida-linked group's first attack against U.S. forces in the East African country, and the military called the security situation "fluid" several hours after the assault.
Five attackers were killed, Kenyan military spokesman Paul Njuguna said.
Al-Shabab, based in neighboring Somalia, claimed responsibility for the assault.
One U.S. serviceman and two contractors with the U.S. Department of Defense were killed in the fighting, according to a statement issued late Sunday by the U.S. Africa Command, or Africom.
The attack on the compound "involved indirect and small arms fire. After an initial penetration of the perimeter, Kenya Defense Forces and U.S. Africa Command repelled the al-Shabaab attack," said the AFRICOM statement. "Reports indicate that six contractor-operated civilian aircraft were damaged to some degree. Manda Bay Airfield is utilized by U.S. forces whose missions include providing training to our African partners, responding to crises, and protecting U.S. interests in this strategically important area."
Al-Shabab claimed that there were 17 U.S. casualties, nine Kenyan soldiers killed and seven aircraft destroyed. The U.S. Africa Command dismissed the al-Shabab claims as exaggerated and said U.S. and Kenyan forces repelled the attack.
Kenya is a key base for fighting al-Shabab, one of the world's most resilient extremist organizations. A large plume of black smoke rose above the airfield Sunday and residents said a car bomb had exploded. Lamu county commissioner Irungu Macharia told The Associated Press that five suspects were arrested and were being interrogated.
An internal Kenyan police report seen by the AP said two fixed-wing aircraft, a U.S. Cessna and a Kenyan one, were destroyed along with two U.S. helicopters and multiple U.S. vehicles at the military airstrip. The report said explosions were heard at around 5:30 a.m. from the direction of the airstrip.
The Kenya Civil Aviation Authority said the airstrip was closed for all operations.
The military's Camp Simba in Lamu county, established more than a decade ago, has under 100 U.S. personnel, according to Pentagon figures. U.S. forces at the adjoining Manda Bay airfield train and give counterterror support to East African partners. A U.S. flag-raising at the camp in August signaled its change "from tactical to enduring operations," the Air Force said at the time.
According to another internal Kenyan police report seen by the AP, dated Friday, a villager that day said he had spotted 11 suspected al-Shabab members entering Lamu's Boni forest, which the extremists have used as a hideout. The report said Kenyan authorities didn't find them.
Al-Shabab has launched a number of attacks inside Kenya, including against civilian buses, schools and shopping malls. The group has been the target of a growing number of U.S. airstrikes inside Somalia during President Donald Trump's administration.
The latest attack comes just over a week after an al-Shabab truck bomb in Somalia's capital killed at least 79 people and U.S. airstrikes killed seven al-Shabab fighters in response.
Last year, al-Shabab attacked a U.S. military base inside Somalia, Baledogle, that is used to launch drone strikes but reportedly failed to make their way inside. The extremist group also has carried out multiple attacks against Kenyan troops in the past in retaliation for Kenya sending troops to Somalia to fight it.
This attack marks a significant escalation of al-Shabab's campaign of attacks inside Kenya, said analyst Andrew Franklin, a former U.S. Marine and longtime Kenya resident.
"Launching a deliberate assault of this type against a well-defended permanent base occupied by (Kenya Defence Forces), contractors and U.S. military personnel required a great deal of planning, rehearsals, logistics and operational capability," he said. Previous attacks against security forces have mainly been ambushes on Kenyan army or police patrols.
The Sunday attack came days after a U.S. airstrike killed Iran's top military commander and Iran vowed retaliation, but al-Shabab is a Sunni Muslim group and there is no sign of links to Shiite Iran or proxies.
"No, this attack was no way related to that incident" in the Middle East, an al-Shabab spokesman told the AP on condition of anonymity for security reasons.
One analyst, Rashid Abdi, in Twitter posts discussing the attack, agreed, but added that Kenyan security services have long been worried that Iran was trying to cultivate ties with al-Shabab.
"Avowedly Wahhabist Al-Shabaab not natural ally of Shia Iran, hostile, even. But if Kenyan claims true, AS attack may have been well-timed to signal to Iran it is open for tactical alliances," he wrote.
But a former member of the U.N. committee monitoring sanctions on Somalia, Jay Bahadur, said in a tweet that "the attack is far more related to AS wanting a do over on their spectacular failure at Baledogle four months ago."
When asked whether the U.S. military was looking into any Iranian link to the attack, U.S. Africa Command spokesman Col. Christopher Karns said only that "al-Shabab, affiliated with al-Qaida, has their own agenda and have made clear their desire to attack U.S. interests."
The al-Shabab claim of responsibility said Sunday's attack was part of its "Jerusalem will never be Judaized" campaign, a rarely made reference that also was used after al-Shabab's deadly attack on a luxury mall complex in Kenya's capital, Nairobi, in January 2019.
Somalia's government, which is fighting al-Shabab with the help of a multinational African force, The Federal Republic of Somalia joins the rest of the world in condemning the cowardly attack that targeted joint Kenyan and U.S forces based at Manda Bay Airfield, Kenya earlier today.
Anna contributed from Johannesburg
Italy's 5-Star implosion may claim party leader Di Maio
by NICOLE WINFIELD, Associated Press / Jan 22, 2020
ROME (AP) —
The head of Italy's 5-Star Movement met with ministers Wednesday amid rumors he would step aside as party leader following a string of parliamentary defections, falling poll numbers and questions about t he movement's future.
Islamic extremists kill Nigerian pastor, attack his hometown
by SAM OLUKOYA, Associated Press / Jan 22, 2020
LAGOS, Nigeria (AP) — Islamic militants in Nigeria have killed a Christian pastor who had pleaded for his life in a video just days earlier, and a human rights activist said Tuesday that other extremists attacked his hometown on the same day.
The Rev. Lawan Andimi was abducted earlier this month when Boko Haram militants attacked the Michika local government area, where he was the chairman of a local chapter of the Christian Association of Nigeria. He was killed on Monday.
by YANAN WANG, Associated Press / Jan 22, 2020
BEIJING (AP) — As a viral outbreak spread from the central Chinese city of Wuhan this week, the ruling Communist Party's central political and legal affairs commission issued a stern warning: "Whoever deliberately delays and conceals reports will forever be nailed to history's pillar of shame."
The proclamation Tuesday signaled both China's growing confidence and its greater awareness of censorship's pitfalls.


The best place to head in a medical emergency is the Beijing United Family Health Center, which has 24-hour emergency services. International SOS is another clinic with a good reputation; it can arrange Medivac. For over-the-counter medicines, Watsons pharmacies can be found all over the city, including in most large shopping centers.
Beijing has different numbers for each emergency service, though staff members often don't speak English. If in doubt, call the U.S. embassy first: staff members are available 24 hours a day to help handle emergencies and facilitate communication with local agencies.
U.S. Embassy. 55 Anjialou Lu, Chaoyang, Beijing, Beijing, 100600. 010/8531–4000; 010/8531–3300; beijing.usembassy-china.org.cn.
Beijing United Family Hospital and Clinics. 2 Jiangtai Lu, Chaoyang, Beijing, Beijing, 100015. 010/5927–7000; 010/5927–7120; www.ufh.com.cn/en.
China-Japan Friendship Hospital. Ying Hua Dong Jie, Heping Li, Beijing, Beijing, 100029. 010/6422–2952; 010/6428–2297; english.zryhyy.com.cn.
Hong Kong International Medical Clinic. Hong Kong Macau Center–Swissotel, 2 Chaoyangmen Bei Da Jie, 9th fl., Chaoyang, Beijing, Beijing, 100730. 010/6553–2288; www.hkclinic.com.
International Medical Center Beijing. Beijing Lufthansa Center, 50 Liangmaqiao Lu, Room 106, Chaoyang, Beijing, Beijing, 100125. 010/6465–1561; www.imcclinics.com.
International SOS. Kunsha Building, 16 Xinyuanli, Wing 1, Suite 105, Chaoyang, Beijing, Beijing, 100027. 010/6462–9112; 010/6462–9100; www.internationalsos.com.
Peking Union Medical College Hospital. 1 Shui Fu Yuan, Dongcheng District, Beijing, Beijing, 100730. 010/6529–5284; www.pumch.cn.
Watsons. Holiday Inn Lido Hotel, Jichang Lu, Chaoyang, Beijing, Beijing, 100015.
Watsons. Sanlitun Village South, 19 Sanlitun Lu, Chaoyang, Beijing, Beijing, 100027. www.watsons.com.cn.


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Kashif Khan
Pakistani Stage And Television Actor.
Kashif Khan is a Pakistani stage and television actor. He was the winner of reality show Comedy Circus paired with Ali Asghar.
His real name is Mohammad Kashif Hasan Khan. Kashif began his career in 1999 as a model. In 2000, he began doing stand-up comedy and stage shows. He has a versatile personality who has appeared in TV serials, films and plays.
His serials include Adha Chula, Adventure time, Zehar Baat. He has acted in plays like Pyar Karna and Lagan. He has generally acted in plays which have a thriller theme. He has also hosted many events. He acted in a Pakistani film titled Amber.
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(WJBK) - A local film company is looking to make a change through their films.
Paris Films is holding a special fundraising event Thursday.
Joining FOX 2 is film producer and actor Paris Jones, along with Andre Ray, the writer and director of Elevated Remedies.


In a world where Hacking, data leaks, data security and cybercrime is more prolific than ever, security needs to be top of everyone's list when reviewing any mobile app product.
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Main / Communication / Phd thesis library information science
Phd thesis library information science
Name: Phd thesis library information science
12 Jul Best Reference Sources for Doctoral and Master's Dissertations and Thesis in Library and Information Science; LIS Ph.D. and MLIS Thesis and Dissertations. General reference databases are listed first which is followed by the repositories and digital libraries of individual countries and universities. Smoothing the information seeking path: Removing representational obstacles in the middle school digital library environment. Ph.D. thesis, University of North. It is mainly based on review of the literature and information collected from Library and Information Science (LIS) schools through personal communication. It provides bibliographic information on the master and doctoral theses of LIS submitted to six Pakistani universities to
6 Aug Download the file form here: Indian_PhD_Thesis_in_LIS Thesis Title in Library and Information Science Name of the Research Scholar. Theses/Dissertations Full-Text Articles in Library and Information Science Providing Information And Public Outreach Across Three U.S. State Archaeology . doctoral research and proliferation of education in Library and information. Science {LIS) in India. . PSC Kumar's biblio raphy listed 41 PhD theses from to.
Library science is mainly concerned with the retrieval and management of information under any form. This discipline has become very complex due to the high. Fair use is often viewed as a topic too difficult for librarians and library staff to engage with and, hence, avoided altogether. However, with the right training. The main goal of this study was to do a heuristic evaluation of the information architecture (IA) of the recently redesigned academic library website of the. Department of Library and Information Science: [8] Collection home page 9- Nov, Women s health information based on web resources an analysis. The Library and Information Science (LIS) education in India may be said to have taken place with the . Table 1: Chronological growth of Ph. D. theses in LIS.
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text:
Home > Administration > Board > Redistricting
General Information about Redistricting
Meeting Dates/Agendas/Minutes/Materials
Maps and Data for Each Draft Redistricting Plan
Members of the public are encouraged to email questions and comments to
How are GJCCD Trustees elected?
There are eight trustees on the Gavilan Joint Community College District Board. Each one represents a specific geographic area, which is called a "trustee area" or election district. Residents of each trustee area vote only for candidates who live in their area. Candidates for each Board seat must live in the trustee area they will represent. This is called a by-district election system.
What is redistricting and why is it needed now?
Every 10 years, a new U.S. Census is conducted. Trustee areas must be relatively equal in total population using the new Census counts. Once new Census data are released, all jurisdictions must evaluate their trustee areas (adopted in 2011 or 2012) to determine if they still have equal-populations using the 2020 counts. If not, trustee area boundaries need to be adjusted to re-balance the trustee area populations. The redistricting process is governed by the U.S. Constitution, federal law, and California law.
When will the new trustee area map be used?
The new trustee areas will be in effect for the November 2022 election.
What criteria are used to create trustee areas?
Federal and state laws require that the trustee areas be nearly equal in population using the most recent Census counts. Some variation is permitted, but the rule of thumb is that the difference between the most- and least-populous election districts should not exceed ten percent of the "ideal" district's population, which is one-eighth of the jurisdiction's total population. We understand that courts have generally accepted this standard for population equality (in school districts).
Federal law also requires that election districts must be drawn to respect protected race/ethnic groups so that their communities are neither divided nor overly concentrated in individual districts. Protected groups are race/ethnic/language groups, including Hispanics, African Americans, Asian Americans, and Native Americans (as well as some other groups).
In addition, the California Elections Code (Section 22000) lists criteria that may be taken into account during the redistricting (and initial districting process):
In adjusting the boundaries of the divisions, the board may give consideration to the following factors: (1) topography, (2) geography, (3) cohesiveness, contiguity, integrity, and compactness of territory, and (4) community of interests of the division.
Who creates the District's maps and how can the public participate in the process?
We have hired professional demographers to draft and revise maps for consideration by the public and the Trustees. Revisions of these draft maps will be made based on feedback received by the Trustees and from the community during the public hearings, Board meetings, or a designated staff member. Residents will be able to provide input about boundaries and suggest criteria for creating boundaries. The districting process will be transparent, and all residents will have the opportunity to suggest map revisions.
What Are "Communities of Interest"?
Recent California legislation for the state legislature, cities, and counties requires that communities of interest be taken into account when drawing plans, and when possible, our demographers will do this, too. A community of interest is a geographical area (such as a neighborhood) that would benefit from being in the same district because of shared interests, views, or characteristics. Downtown areas, historic districts, and housing subdivisions are a few examples of areas that would be communities of interest (there are many more).
Populations with similar demographic characteristics are sometimes considered communities of interest. These characteristics include the population's race/ethnic composition, the language spoken at home, the area's median income (and other socioeconomic characteristics), and the type of housing (for example, those in a public housing development or retirement community).
Defined regions like cities, school districts, school attendance areas may sometimes be considered communities of interest. Geographic characteristics can identify communities, such as those on one side of a highway, around a park, or another publicly-recognized region.
Has the pandemic affected the redistricting process?
Because of the COVID pandemic, the Census Bureau is about six months behind schedule in releasing data. This means that we have about half the amount of time to redistrict as we did in the past. As a result, timelines must be very short.
We may provide virtual meetings rather than in-person meetings, or have "hybrid" meetings, in which some people are in-person while others are available virtually.
What data are used when drawing maps?
Plans for election district boundaries will be based on the total population as counted in the 2020 Census, as well as criteria required by federal and state law.
In this round of redistricting, 2020 Census counts will be adjusted by the California Statewide Database (SWDB), which will assign prison inmates to their pre-incarceration home addresses. Congress, city councils, and county boards of supervisors must use these adjusted numbers. Although GJCCD is not required to use the adjusted data, which will be available 4-6 weeks after the Census Bureau releases its redistricting database. Because timelines are already shortened as a result of the pandemic, we may begin redistricting using the unadjusted Census counts for drawing boundaries and then switch to adjusted counts 4-6 weeks later, when the SWDB releases its database.
Supplemental information on the characteristics of the population of each trustee area will be gathered from the American Community Survey (ACS), the U.S. Census Bureau's ongoing survey of the nation's population. Data from this survey are used for several purposes during redistricting to help identify communities of interest, as well as for other purposes.
Who decides which map to adopt?
The Board of Trustees will adopt an ordinance establishing election district boundaries. The county Registrars of Voters will then adjust precinct boundaries before the filing deadline for the November 2022 trustee election.
When will the process begin and end?
We expect there to be an orientation for Trustees and members of the public in October or November. One or more draft plans will be provided by our demographers before the Thanksgiving holidays. Members of the public are encouraged to provide comments about the draft map(s) and/or indicate other ways they suggest trustee areas be drawn.
How long will the boundaries be in place?
By law, election district boundaries must be evaluated after each decennial census. The 2030 U.S. Census redistricting population counts will be released in 2031. If the trustee areas adopted in 2022 still have equal total population counts, the boundaries will not need to be adjusted. If the total populations are not equally distributed, the trustee area boundaries will need to be adjusted so that the 2030 population is distributed evenly in the eight trustee areas.
What will happen to current members of the Board of Trustees?
The current Board Members will continue in office until the expiration of their terms and their successors are elected. The first elections using the new boundaries will be in November 2022. Because Board elections are staggered (three or four trustees elected in 2022 and the rest elected in November 2024), the new plan will be completely implemented in 2024. If current Trustees' terms end in 2024, they will continue to sit on the Board even if they live in the same election district as a Board member elected in 2022.
What is the California Fair Maps Act of 2019 and does it apply to our District?
The California Fair Maps Act of 2019 (AB 849) applies to cities and counties, but not to school districts and special districts. However, GJCCD might use some of these guidelines when it is reasonable to do so.
The Fair Maps Act lists the following criteria, in order of priority, that cities and counties should (or should not) use during plan drawing, after federal legal requirements are met:
Geographic contiguity
Communities of interest are respected.
Other governmental boundaries (e.g., cities, school districts, established neighborhoods) may be used for election district boundaries.
Boundaries are easily identifiable and understandable.
Geographic compactness
Political parties are not to be considered.
There is no specific mention of incumbency, except that incumbency is not a community of interest.
In addition, the FAIR MAPS Act requires that cities and counties:
Hold hearings so that members of the public can provide input on the placement of boundaries and on proposed boundary maps.
Take steps to encourage the residents of the local jurisdiction to participate in the redistricting process.
Redistricting Project


Two weeks ago I had the pleasure of spending the evening with these two amazing women! We started our session at the beautiful Charmlee Wilderness Park and finished at the iconic Leo Carrillo cliffs! Camille and Anastasia didn't meet until Stasia moved to Los Angeles from Chicago in 2013. They were both working as nurses at the same hospital, but didn't start dating until after they attended a Third Eye Blind concert with a group of their mutual friends! Ending their incredible night at one of my personal favorites spots, Carney's, they've been inseparable ever since. 🙂 Next up, these two are off to Kauai in April to say there I Do's!