text:
Frontiers in Plant Science
Agnieszka Ludwików
Adam Mickiewicz University, Poland
Biswapriya B. Misra
Wake Forest Baptist Medical Center, United States
Taras P. Pasternak
University of Freiburg, Germany
Front. Plant Sci., 05 November 2015 | https://doi.org/10.3389/fpls.2015.00956
Endogenous target mimics down-regulate miR160 mediation of ARF10, -16, and -17 cleavage during somatic embryogenesis in Dimocarpus longan Lour
Yuling Lin, Zhongxiong Lai*, Qilin Tian, Lixia Lin, Ruilian Lai, Manman Yang, Dongmin Zhang, Yukun Chen and Zihao Zhang
Institute of Horticultural Biotechnology, Fujian Agriculture and Forestry University, Fuzhou, China
MicroRNA160 plays a critical role in plant development by negatively regulating the auxin response factors ARF10, -16, and -17. However, the ways in which miR160 expression is regulated at the transcriptional level, and how miR160 interacts with its targets during plant embryo development, remain unknown. Here, we studied the regulatory relationships among endogenous target mimics (eTMs), and miR160 and its targets, and their involvement in hormone signaling and somatic embryogenesis (SE) in Dimocarpus longan. We identified miR160 family members and isolated the miR160 precursor, primary transcript, and promoter. The promoter contained cis-acting elements responsive to stimuli such as light, abscisic acid, salicylic acid (SA) and heat stress. The pri-miR160 was down-regulated in response to SA but up-regulated by gibberellic acid, ethylene, and methyl jasmonate treatment, suggesting that pri-miR160 was associated with hormone transduction. Dlo-miR160a, -a∗ and -d∗ reached expression peaks in torpedo-shaped embryos, globular embryos and cotyledonary embryos, respectively, but were barely detectable in friable-embryogenic callus. This suggests that they have expression-related and functional diversity, especially during the middle and later developmental stages of SE. Four potential eTMs for miR160 were identified. Two of them, glucan endo-1,3-beta- glucosidase-like protein 2-like and calpain-type cysteine protease DEK1, were confirmed to control the corresponding dlo-miR160a∗ expression level. This suggests that they may function to abolish the binding between dlo-miR160a∗ and its targets. These two eTMs also participated in 2,4-D and ABA signal transduction. DlARF10, -16, and -17 targeting by dlo-miR160a was confirmed; their expression levels were higher in friable-embryogenic callus and incomplete compact pro-embryogenic cultures and responded to 2,4-D, suggesting they may play a major role in the early stages of longan SE dependent on 2,4-D. The eTMs, miR160, and ARF10, -16, and -17 exhibited tissue specificity in 'Sijimi' longan vegetative and reproductive organs, but were not significant negatively correlated. These results provide insights into the possible role of the eTM-miR160-ARF10-16-17 pathway in longan somatic embryo development.
MicroRNAs, a conserved class of single-stranded non-coding RNAs of approximately 21 nt in length, are derived from pri-miRNAs containing imperfect stem-loop secondary structures (Jones-Rhoades et al., 2006). These hairpin RNAs, which are known as pre-miRNAs genes, are transcribed by RNA polymerase II in plants, processed by DICER- LIKE1 to produce an miRNA:miRNA∗ duplex in the nucleus, and transported to the cytoplasm (Lee et al., 2004; Jones-Rhoades et al., 2006). The mature miRNA recognizes target mRNA sites through perfect or near perfect complementarity, and negatively regulates mRNA expression by cleaving targeted mRNAs (Llave et al., 2002) or repressing translation (Gandikota et al., 2007). miRNAs play vital roles in a range of developmental processes (Carrington and Ambros, 2003; Jones-Rhoades et al., 2006). The expression of miRNA genes is tightly controlled. Recently, a novel mechanism involving eTMs was discovered, whereby miRNA–mRNA duplexes with a large bulge around the cleavage site actually sequester miRNAs, resulting in inhibition of miRNA activity (Franco-Zorrilla et al., 2007). eTMs have been used as a valuable tool to understand the functions of several miRNA families in plants (Wu et al., 2013).
A highly conserved miRNA family, miR160, negatively regulates ARF10 (Liu et al., 2007), -16 (Wang et al., 2005), and -17 (Mallory et al., 2005) of the repressor ARF family, and plays a critical role in maintaining the process of seed germination and the normal developmental programs of embryos, roots, stems, leaves, siliques, and floral organs. An Arabidopsis loss-of-function mutation in miR160a caused various embryonic defects, suggesting that miR160 may control embryo development (Liu et al., 2010b). Ectopic expression of miR160 results in auxin hypersensitivity, cytokinin hyposensitivity, and inhibition of symbiotic nodule development in soybean (Turner et al., 2013), and decreases seed dormancy in Arabidopsis (Liu et al., 2013). The over-expression of artificial target mimics for miR160 resulted in plants of a smaller size with serrated and upward-curled leaves (Todesco et al., 2010; Yan et al., 2012). Over-expression of eTMs of miR160 also caused smaller and serrated leaves in transgenic plants (Wu et al., 2013), suggesting that eTMs play a key role in plant development by inhibiting miRNA activity. However, to date, analysis and identification of eTMs for miR160 has rarely been performed in plants (Wu et al., 2013; Shuai et al., 2014).
Dimocarpus longan Lour., also called longan or dragon eye, is a tropical tree of the soapberry family (Sapindaceae) that produces edible fruit. As a traditional medicine, longan fruit is used to enhance memory (Park et al., 2010), promote blood metabolism, relieve insomnia, and prevent amnesia (Yi et al., 2011). Longan seed extracts, which contain high levels of antioxidant polyphenolic compounds (Chung et al., 2010) and polysaccharides (Zheng et al., 2010), are capable of inducing apoptosis in various colorectal cancer cells. Previous studies have shown that longan's embryo development status influences its seed size, fruit quality, fruit set and yield (Liu et al., 2010a; Lai and Lin, 2013; Lin and Lai, 2013a,b). However, because of the extreme genetic heterozygosity exhibited by this species and the difficulty of sampling early embryos, advances in understanding of the regulation of longan embryo development have been limited.
Somatic embryogenesis, which resembles zygotic embryogenesis, is an essential component of plant cell differentiation and embryo development. The longan SE system has been used widely as a model system to investigate in vitro and in vivo regulation of embryogenesis in plants (Lai et al., 2010; Lin and Lai, 2010). In our previous study, we found that miR160a was barely detectable during the early stages of longan SE, but highly expressed during the heart- and torpedo-shaped embryonic stages (Lin and Lai, 2013b). Previous reports have suggested that larch and Brassica napus miR160s might play a regulatory role during cotyledonary embryo development (Zhang et al., 2012; Huang et al., 2013). However, it is not clear how post-transcriptional regulation of ARF10, -16, and -17 by miR160 regulates plant embryo development or SE, particularly in longan.
The aim of this study was to investigate the regulatory networks of eTMs and miR160-mediated regulation of ARF10, -16, and -17 in longan, especially during SE. Here, we identified and examined the expression of mature miR160 molecules, the miR160 primary transcript and its promoter, eTMs, and homologs of the target genes ARF10, -16, and -17 for miR160. Our results suggest possible general regulation by eTM down-regulation of miR160 cleavage of ARF10, -16, and -17 during SE in longan.
Tissue Samples
Synchronized embryogenic cultures at different developmental stages from D. longan 'Honghezi' (Fujian, China), consisting of EC, ICpEC, GE, HE, TE, and CE, were generated as detailed in Lai and Chen (1997).
The EC was pre-cultured on Murashige and Skoog (MS) medium containing 2,4-D (1.0 mg/L) for 20 days, and then sub-cultured in 50 mL of liquid MS medium containing 2,4-D (0, 0.5, 1.5, or 2 mg/L), SA (75 μM), MeJA (50 μM), ET (30 ppm), GA3 (35 μM), or ABA (100 μM) in a rotary shaker at 150 rpm for 24 h. These cultures were all kept in the dark.
Dimocarpus longan 'Sijimi' samples, including roots, leaves, floral buds, flowers, young fruits, mature fruits, pericarp, pulp and seeds, were collected from the experimental fields of Fujian Academy of Agricultural Science in Putian. All materials from at least six rootstock plants of the same cultivar were combined.
The synchronized embryogenic cultures, EC cultures treated with different hormones, and 'Sijimi' samples were collected and stored at -80°C for subsequent analyses.
Database Searching for miR160 Members of Longan
Previously published small RNA data (BioSample accession SAMN04120614, Bio-Project ID PRJNA297248) (Lin and Lai, 2013b), generated using various synchronized embryogenic cultures from the longan 'Honghezi' variety, was used to identify miR160 family members. The potential miR160 members were searched using the miRBase server (Release 21: June 2014). Multiple alignment analysis of miR160 members was performed using DNAMAN ver. 6.0 (Lynnon Biosoft Corp, Pointe-Claire, QC, Canada).
Pri-miR160 3′ and 5′ RACE Experiments
To determine whether the miR160 members were present in the D. longan genome, the full-length cDNA sequence of pri-miR160 was cloned by RT-PCR and 3′/5′ RACE PCR from the EC and mixed cDNA. The EC cDNA for RT-PCR of the pre-miR160 conserved regions was synthesized using a RevertAid First Strand cDNA Synthesis Kit (Fermentas, Thermo Fisher Scientific, Waltham, MA, USA). The mixed cDNA for pri-miR160 3′/5′ RACE PCR was synthesized from mixed total RNA (EC, ICpEC, GE and HE) using a GeneRacerTM Kit (Invitrogen, Corporation, Carlsbad, CA, USA), following the manufacturer's instructions.
The amplification of the full-length cDNA of pri-miR160 was carried out by 3′/5′ RACE PCR from the longan mixed cDNA, and its genomic sequence was cloned from the EC DNA template. The sequences of all primers used are provided in Table 1.
TABLE 1. Gene names and primer sequences.
To determine the phylogeny of the miR160 gene, we downloaded all precursor sequences of plant miR160s from miRBase release 21. Phylogenetic analysis was performed according to the neighbor-joining method (Saitou and Nei, 1987) using MEGA 5.02 (Kumar et al., 2004), with 1000 bootstrap replicates.
miR160 Promoter Cloning and cis-Acting Element Analysis
To study the molecular mechanism underlying the transcriptional regulation of miR160, genome walking was performed using TAIL-PCR (Takara, Japan) to obtain the miR160 promoter. The promoter was cloned by three nested PCR amplifications from the longan EC DNA template with the same forward primer AP2 and specific reverse primers. The primer sequences can be found in Table 1. Cis-acting element analysis of the miR160 promoter was carried out using the PlantCARE database (Lescot et al., 2002).
Prediction of eTMs for the miR160 Family in D. longan
First, the longan EC transcriptome database (SRA050205) (Lai and Lin, 2013) was used to search for sites in the cDNA sequences that were reverse complements of the miR160 family, and the tool TAPIR was used to search for eTMs according to criteria described previously (Bonnet et al., 2010). The cDNAs that satisfied the above criteria were considered to be target mimic candidates.
Modified 5′ RLM-RACE
To determine the internal cleavage sites in the ARF10, -16, and -17 mRNAs, a modified 5′ RLM-RACE experiment was performed using a GeneRacer kit (Invitrogen). Briefly, total RNAs from equal mixtures of EC, GE, HE, and TE were ligated to a 5′ RACE RNA adapter. The experiment essentially followed the manufacturer's instructions. The PCR amplification used GeneRacer 5′ primers and gene-specific primers (Table 1). The resulting PCR products were gel-purified, cloned, and sequenced.
Gene Expression Assays
Total RNAs were isolated from the nine synchronized embryogenic cultures described above using the TRIzol Reagent kit (Invitrogen). cDNAs for miRNA and mRNA quantification were synthesized using a One Step PrimeScript® miRNA cDNA Synthesis Kit (Takara Code, D350A), and PrimeScriptTM Perfect Real Time RT Reagent Kit (TaKaRa Code, DR037A), respectively.
Quantitative PCR assays of gene expression were performed using a LightCycler 480 qPCR instrument (Roche Applied Science, Switzerland) and SYBR premix (Takara). All reactions were performed in triplicate. The expression levels of the miR160s, their targets (ARF10, -16, -17), and the eTMs were quantified using internal standards (Lin and Lai, 2010, 2013c). Statistical analysis was performed using SPSS 19. Gene names and primer sequences are provided in Table 2.
TABLE 2. Gene names and primer sequences for qPCR analysis.
Analysis of the miR160 Family Members from a Longan Small RNA Dataset
The miR160 family, comprising three members (ath-miR160a–c) in Arabidopsis and six members (osa-miR160a–f) in Oryza sativa, was first identified in plants (Reinhart et al., 2002). By searching a longan small RNA dataset, we found that the miR160 family was represented by 12 members with different read counts (Figures 1A–C). The 12 potential miR160 members were then compared against the miRBase database (Release 21), which revealed that they were distributed into two isoforms. One was identical to ath-miR160a, including ten similar variants; the other was identical to osa-miR160d, including two variants. We named these two isoforms dlo-miR160a and dlo-miR160d, respectively. However, variants similar to miR160b and miR160c were not found. Therefore, there are at least two miR160 isoforms in the D. longan genome. According to the Blast alignment results, of these 12 variants, only two mature miRNAs were excised from the 5′ arm of the hairpin, the others were located in the 3′ arm of the hairpin.
FIGURE 1. Alignment of Dimocarpus longan miR160 members. (A) miR160a; (B) miR160a∗; (C) miR160d∗. ATH, Arabidopsis thaliana (miRBase No. MI0000190); BRA, Brassica rapa (miRBase No. MIMAT0010155); PTC, Populus trichocarpa (miRBase No. MIMAT 0022890); OSA, Oryza sativa (miRBase No. MIMAT0022854); DLO, Dimocarpus longan.
Identification of the miR160 Gene Sequence in Longan
To find the miR160 gene and determine its structure and length in the D. longan genome, the full-length cDNA sequence of pri-miR160 was obtained by 3′/5′-RACE. Two precursors with lengths of 80 and 82 bp were obtained by RT-PCR. Alignment analysis showed that these two precursors were highly homologous except for 13 nucleotide differences (Figure 2A). To determine the hairpin structure of the miR160 precursors and the positions of the miR160 members, secondary structure analysis of the precursors was conducted using the DNAMan6.0 software. The two miR160 precursors both formed the classic stem-loop structure, and minimum free energy (MFE) analysis indicated that the stabilities of their secondary structures were -47.1 and -39.5 kcal/mole, respectively (Figure 2B). By comparing these two precursors and the mature miR160 sequences, dlo-miR160a-5p variants were found that exactly matched the 5′ arms of both precursors, while dlo-miR160a-3p and -d-3p variants were detected in the 3′ arms of the two precursors, respectively. The sequence that exactly matched the dlo-miR160a-3p variants was named pre-miR160a; the other one was identical to dlo-miR160d-3p and was named pre-miR160d (Figure 2B). Phylogenetic analysis (Figure 2C) also suggested that the pre-miR160a sequence in D. longan was homologous to pre-miR160 sequences in Arabidopsis (miR160a), Populus trichocarpa (miR160b, c), and Vitis vinifera (miR160e). Pre-miR160d appeared to be closely related to V. vinifera miR160d.
FIGURE 2. Separate primary MIR160 transcripts contain dlo-miR160a/a∗ and -160d∗. (A) Longan miR160 precursors. (B) Hairpin structures of the longan miR160 precursors containing dlo-miR160a/a∗ and -160d∗. (C) Neighbor-joining phylogenetic tree of pre-miR160 sequences in plants. Numbers in the tree represent bootstrap values. ptc, Populus trichocarpa; vvi, Vitis vinifera; dlo, Dimocarpus longan; ath, Arabidopsis thaliana; osa, Oryza sativa.
Next, 5′ and 3′ RACE analyses for the pri-miR160 transcripts were carried out. The full-length sequence was 441 bp (GenBank accession No. KJ372214), with the typical poly(A) tail in the 3′ UTR, and the first nucleotide (transcription initiation site) of the full-length cDNA was A. BLAST analysis of the pri-miR160 showed similarities with existing pri/pre-miR160s in Manihot esculenta, P. trichocarpa, Gossypium hirsutum, and Arabidopsis thaliana. The corresponding pri-miR160 genomic DNA sequence, which comprised 424 bp and contained one 100 bp intron, was also obtained (GenBank accession No. KJ372217). The sequence at the exon–intron junctions was consistent with the eukaryotic GT–AG rule.
Isolation of the miR160 Promoter in Longan
To further understand the regulatory role of the miR160 gene at the transcriptional level, a 957 bp 5′-flanking sequence of the pri-miR160 was cloned from longan EC (GenBank accession No. KJ372217). PlantCARE (Lescot et al., 2002) was used to search for cis-acting elements in the pri-miR160 promoter. The results revealed the presence of conserved TATA and CAAT boxes. Many putative cis-elements associated with responses to light (BOX-4, Box II, G-BOX, GAG-motif, GATA-motif and Sp1), ABA (ABRE), SA (TCA-element) and environmental stresses (heat stress-response element, HSE) were found in the promoter. In addition, an anaerobic response element (ARE) that is essential for anaerobic induction, a Skn-1_motif that is required for endosperm expression, a circadian element involved in circadian control, and four elements of unknown function were also present in the promoter (Figure 3). These results prompted us to carry out further experiments to identify the response of the miR160 gene to various environmental stimuli.
FIGURE 3. 5′ flanking sequence of pri-miR160 in D. longan. The bold A is the transcription start site (TSS). Cis-acting regulatory elements are underlined and shaded in gray; their orientations are shown by arrows.
Transcriptome-wide Prediction of eTMs of the miR160 Family in Longan
To date, an analysis of eTMs for miR160 has not been performed in longan. Here, transcriptome-wide computational prediction of eTMs for miR160 was performed. Four potential eTMs were identified, including an unknown mRNA (Unigene11608), a nitrate transporter family protein (Unigene56365), glucan endo-1,3-beta- glucosidase-like protein 2-like (Unigene64827), and calpain-type cysteine protease DEK1 (Unigene66891) (Figure 4). By contrast, two and eleven non-coding eTMs for miR160 were predicted in Arabidopsis and rice, respectively (Wu et al., 2013). The four potential eTMs all contained a three-nucleotide insertion between the nucleotides interacting with positions 10 and 11 of the corresponding miRNA, and the mimic sites of Unigene11608 and Unigene56365 were on the sense strand of the 3′ UTR and 5′ UTR, respectively, while the other two mimics were located on the antisense strand of the CDS (coding sequence) region (Figure 4). These results suggested that the miR160 family might also be controlled by eTMs in D. longan.
FIGURE 4. Predicted eTMs for the miR160 family in longan. (+), mRNA sense strand; (-), mRNA antisense strand; RPKM, reads per kilobase per million mapped reads; size, unigene length. (A) Unigene11608: an unknown mRNA; (B) Unigene56365: a nitrate transporter family protein; (C) Unigene64827: glucan endo-1,3-beta- glucosidase-like protein 2-like; (D) Unigene66891: calpain-type cysteine protease DEK1.
ARF Transcripts are Cleaved by miR160 During Longan SE
In Arabidopsis, miR160 targets ARF10, -16, and -17, and its regulation of these transcripts appears to be important in seed germination, and shoot and root development (Mallory et al., 2005; Wang et al., 2005; Liu et al., 2007); however, its role in longan SE remains unknown. The mRNA sequences of the D. longan ARFs DlARF10 (KJ410234), -16 (KJ410235), and -17 (KJ410237) were extracted from GenBank to search for possible miR160 cleavage sites. Alignment analysis showed that DlARF10, -16, and -17 shared high amino acid sequence similarities. All contained a conserved family domain and a B3 domain, while DlARF17 was distinguished by its poorly conserved C-terminal domain (Figure 5), which was consistent with the ARFs identified from Arabidopsis (Wang et al., 2005).
FIGURE 5. Cleavage site mapping of dlo-miR160a target genes. The mRNA sequences of DlARF10, -16, and -17 are aligned with dlo-miR160a. Numbers indicate the fraction of cloned PCR products terminating at different positions. The B3 DNA binding domain (B3), Auxin_resp, and AUX_IAA are highlighted in green, red, and blue, respectively, in the ARF10, -16, and -17 proteins. The dlo-miR160a complementary sequence in the DlARF10, -16, and -17 mRNAs and the corresponding amino acid sequences are shown.
Fragments of the DlARF10, -16, and -17 mRNAs were detected from longan embryogenic tissues and were determined to be cleaved by miR160 using a modified form of RLM-RACE. The miR160a target sites in the DlARF10, -16, and -17 mRNAs were located at positions corresponding to the amino acid sequences GMQGAWH, GIQGARH, and GMQGARQ, respectively. The miR160a sequence showed high complementarity to these regions in DlARF10, -16, and -17, with only one or two mismatched bases (Figure 5). To determine the tissues in which miR160a regulates the ARF10, -16, and -17 mRNAs, we searched for cleaved fragments of all three genes in longan EC cDNA and mixed cDNA samples. Fragments of the three genes were not detected in the EC samples, but were found in the mixed cDNA sample, indicating the presence of cleaved products in the mixed cDNA, but not in EC. These results clearly indicated that miR160a cleaved the ARF10, -16, and -17 mRNAs during longan SE, confirming that miR160-ARF10-16-17 signal transduction operates in longan, similarly to Arabidopsis.
Confirmation of an eTM-miR160-ARF10-16-17 Regulatory Pathway During Longan SE
To determine experimentally whether the potential dlo-miR160 family members obtained from the longan small RNA data were indeed expressed, and whether the predicted eTMs function to abolish the binding between miR160 and its targets (ARF10, -16, -17), we investigated their expression patterns during SE in D. longan (Figures 6 and 7).
FIGURE 6. Relative expression of eTMs (Unigene64827/66891), the dlo-miR160 family, miR160 targets and pri-miR160 during longan SE. (A–D) The expression levels were normalized to the reference genes DlFSD1a, EF-1a and eIF-4a. Unigene64827: glucan endo-1,3-beta-glucosidase-like protein 2; Unigene66891: calpain-type cysteine protease DEK1 family. Samples: EC, friable-embryogenic callus; ICpEC, incomplete compact pro-embryogenic cultures; GE, globular embryos; HE, heart-shaped embryos; TE, torpedo-shaped embryos; CE, cotyledonary embryos. The y-axes represent the relative expression values; the x-axes represent longan SE samples. Asterisks (∗) and different lowercase letters above the bars indicate a statistically significant difference, and identical lowercase letters denote no significant difference among different embryogenic cultures (P < 0.05).
FIGURE 7. Morphology of the six sequential developmental stages of longan SE. (A) Friable-embryogenic callus (EC); (B) incomplete compact pro-embryogenic cultures (ICpEC); (C) globular embryos (GE); (D) heart-shaped embryos (HE); (E) torpedo-shaped embryos (TE); (F) cotyledonary embryos (CE). The bars are 50 μm in (A), 100 μm in (B–D), and 500 μm in (E,F).
The expression levels of dlo-miR160a, -a∗, and -d∗ were investigated during D. longan SE (Figures 6A–C). Members of the dlo-miR160 family exhibited different temporal and spatial expression. Dlo-miR160a and -a∗ both exhibited tissue-specific expression and showed similar expression patterns; both were barely detectable in EC. The abundance of dlo-miR160a increased during the ICpEC, GE and HE stages and reached a peak in the TE stage, while dlo-miR160a∗ showed its highest expression in GE. In addition, dlo-miR160d∗ expression was lowest in EC, but continually increased in ICpEC-GE-TE, reaching its peak in the CE stage. These results showed that different members of the dlo-miR160 family might have different and separate regulatory effects on longan SE. Furthermore, the expression levels of the mature miR160s and pri-miR160 were compared. Pri-miR160 showed a similar expression pattern to dlo-miR160a∗, but had some differences compared with the expression levels of dlo-miR160a and -d∗ (Figure 6D).
To examine whether the four predicted eTMs for dlo-miR160a∗ were indeed transcribed, and to test whether these eTMs control miR160 expression, we validated their expression during longan SE. The unknown mRNA (Unigene11608) and nitrate transporter family protein (Unigene56365) were not detected in the six tissues. However, endo-1,3-beta-glucosidase-like protein 2-like (Unigene64827) and calpain- type cysteine protease DEK1 (Unigene 66891) showed great variation among the tissues (Figure 6C). Both were highly expressed in the HE and CE stages, and weakly expressed in EC, suggesting that they might play potential roles in the late stages of SE. Their expression patterns were largely reciprocal to that of dlo-miR160a∗, especially at the GE to CE stages (Figure 6C). These results showed that the predicted eTMs (endo-1,3-beta-glucosidase-like protein 2-like and calpain-type cysteine protease DEK1) may indeed function to abolish the binding between dlo-miR160a∗ and its targets.
DlARF10, -16, and -17 targeting by dlo-miR160a members was confirmed by RLM- RACE during longan SE, and their expression levels were further examined in six tissues (Figure 6A). DlARF10 and -16 both reached their peaks in the EC stage, while DlARF17 expression was highest in the ICpEC stage. DlARF10 and -17 expression was lowest in the GE stage, and DlARF16 was expressed weakly in the CE stage. As expected, their expression was mainly inversely proportional to that of dlo-miR160a (Figure 6).
Taken together, these results confirmed the eTM-miR160-ARF10-16-17 regulatory pathway might play a potential role during the SE process in D. longan.
2,4-D Controls the Levels of the eTMs, miR160, and ARF10, -16, and -17
Auxin plays major roles in embryo development. To investigate whether the miR160 level and eTM-miR160-ARF10-16-17 signal transduction were controlled by exogenous auxin, they were analyzed in longan EC grown for 24 h on media containing different concentrations of 2,4-D (an auxinic herbicide). The results showed that the 2,4-D concentration affected the eTM, miR160, and ARF10, -16, and -17 levels (Figure 8).
FIGURE 8. Auxin control of eTM, miR160, and ARF10/-16/-17 expression levels. (A,B) The expression levels were normalized to the reference genes DlFSD1a, EF-1a and eIF-4a. Asterisks (∗) and different lowercase letters above the bars indicate a statistically significant difference, and identical lowercase letters denote no significant difference among different embryogenic cultures (P < 0.05).
Pri-miR160 accumulated at 0.5 mg/L 2,4-D after 24 h of treatment compared with auxin-free medium under the same conditions, but decreased significantly at higher concentrations (Figure 8), substantiating the participation of miR160 in the 2,4-D signal pathway. The expression patterns of the eTMs (endo-1,3-beta-glucosidase-like protein 2-like and calpain-type cysteine protease DEK1) resembled those of DlARF16 and -17 targeted by dlo-miR160a under the tested concentrations of 2,4-D (Figure 8). Both were expressed at lower levels at 0.5 mg/L 2,4-D compared with auxin-free medium, increased at 1.5 mg/L, and decreased at 2.0 mg/L, which was largely reciprocal to pri-miR160 expression. This result demonstrated that the presence of auxin in the medium affected the levels of the eTMs (endo-1,3-beta-glucosidase-like protein 2-like and calpain-type cysteine protease DEK1), and in turn, miR160- mediated regulation of DlARF16 and -17 in longan EC. However, the level of DlARF10 increased as the 2,4-D concentration increased (0.5–1.5 mg/L), but decreased at 2.0 mg/L, which was not the reverse of pri-miR160. This suggested that DlARF10 responds to 2,4-D in a positive manner, but might be out of miR160's control.
Taken together, these results clearly demonstrated that 2,4-D controlled the levels of the eTMs (endo-1,3-beta-glucosidase-like protein 2-like and calpain-type cysteine protease DEK1), and in turn, miR160-mediated regulation of DlARF16 and -17 in longan. However, it is not clear whether auxin affects the eTM levels directly.
ABA Regulates eTM-miR160 Signal Transduction
A previous study showed that miR160-ARF10 plays an important role in ABA-auxin crosstalk in seed germination and post-embryonic developmental programs (Liu et al., 2007). To test whether ABA treatment also affected miR160 expression and eTM-MIR160 signal transduction in longan, longan EC was grown for 24 h on medium containing 100 μM ABA and analyzed. The results showed that ABA treatment affected the eTM and miR160 levels (Figure 9). After ABA treatment, the levels of the eTMs (endo-1,3-beta-glucosidase-like protein 2-like and calpain-type cysteine protease DEK1) were slightly decreased compared with the control, while pri-miR160 expression was elevated after 24 h. Thus, the increased expression of pri-miR160 in longan EC after ABA treatment might be caused by decreased expression of the eTMs (endo-1,3-beta-glucosidase-like protein 2-like and calpain- type cysteine protease DEK1), or through the ABRE element in its 5′ regulatory region.
FIGURE 9. Exogenous ABA treatment affects the eTM and miR160 expression levels. The expression levels were normalized to the reference gene EF-1a. Unigene64827: glucan endo-1,3-beta-glucosidase-like protein 2, Unigene66891: calpain-type cysteine protease DEK1 family. An asterisk (∗) indicates a statistically significant difference between the control (C) and hormone treatments (P < 0.05). The y-axes represent relative expression values; the x-axes represent hormone concentration.
Expression Changes of Pri-miR160 in Response to Hormone Treatments
To further analyze the possible functions of miR160 in phytohormone signaling pathways, the expression of pri-miR160 in response to hormonal stimuli was investigated. The pri-miR160 expression level was greatly increased in response to GA3 (35 μM), ET (30 ppm), and MeJA (50 μM) treatments, but was slightly decreased after 24 h of SA (75 μM) treatment (Figure 10). Thus, GA3, ET, and MeJA up-regulate the expression of miR160, while SA down-regulates its expression in longan EC, possibly through related cis-elements in its promoter region. However, only an SA-responsive cis-element (TCA-element) was predicted in the promoter region. These results suggest that miR160 may also mediate these hormone signal interactions.
FIGURE 10. Expression analysis of pri-miR160 in response to exogenous hormone treatments. The expression levels were normalized to the reference gene EF-1a. An asterisk (∗) indicates a statistically significant difference between the control (C) and hormone treatments (P < 0.05). The y-axes represent relative expression values; the x-axes represent hormone concentration.
Expression of eTMs, miR160, and ARF10, -16, and -17 in 'Sijimi' Tissues
To get more information about the characteristics of the eTM, miR160, and ARF10, -16, and -17 accumulation patterns in other tissues, their expression in D. longan Lour. cv. Sijimi was studied (Figures 11 and 12). 'Sijimi' is unique cultivar of longan that blossoms and bears fruit throughout the year. A variety of tissues (Figure 12), including roots (R), mature leaves (L), floral buds (FB), flowers (F), young fruits (YF), mature fruits (MF), pericarp (P1), pulp (P2) and seeds (S), were used to assay RNA accumulation.
FIGURE 11. Differential expression analysis of eTMs, miR160, and DlARF10, -16, and -17 in vegetative and generative tissues of 'Sijimi' longan. (A–F), qPCR analysis of the expression levels of the eTMs, miR160, and DlARF10/-16/-17. Unigene64827: glucan endo-1,3-beta-glucosidase-like protein 2, Unigene66891: calpain-type cysteine protease DEK1 family. The expression levels were normalized to the reference genes DlFSD1a, EF-1a, and eIF-4a. Samples: roots (R), leaves (L), floral buds (FB), flowers (F); young fruit (YF), mature fruit (R), pericarp (P1), pulp (P2), and seeds (S).
FIGURE 12. Morphology of vegetative and generative tissues of 'Sijimi' longan. (A) Roots (R); (B) leaves (L); (C) flowers (F) and floral buds (FB); (D), young fruit (YF); (E) mature fruit (R) and pericarp (P1, arrow); (F) pulp (P2, arrow), and seeds (S). Bars are 8 mm.
We examined the expression patterns in all tissues and found that the eTMs, miR160 and ARF10, -16, and -17 exhibited tissue specificity (Figures 11A–F). All the genes were highly expressed in roots, and showed the lowest expression levels in mature leaves, except for calpain-type cysteine protease DEK1 (Unigene 66891), which was least expressed in mature fruits. These observations indicated that eTM-miR160-ARF10- 16-17 signal transduction might affect 'Sijimi' root development. In addition, high expression levels were observed for pri-miR160, endo-1,3-beta-glucosidase-like protein 2-like (Unigene 64827) and DlARF17 in seeds, calpain-type cysteine protease DEK1 (Unigene 66891) and DlARF10 and -16 in pulp, DlARF10 in flowers, and DlARF16 in floral buds and pericarp. Furthermore, the expression of pri-miR160 in young fruits and pulp, DlARF10 and -16 in young fruits and mature fruits, and DlARF17 in mature fruits was very low or undetectable. These data suggested eTM-miR160-ARF10-16-17 signal transduction plays an extensive role in 'Sijimi' development, but there was no significant negative correlation among the levels of eTM, miR160 and DlARF10, -16, and -17 mRNA accumulation in the organs analyzed.
Unique Size and Structure of longan miR160
Plant miRNAs are usually encoded by small gene families of up to 14 members (Parizotto et al., 2004), similar to protein-coding genes. The miR160 family in plants includes three members in Arabidopsis, six members in O. sativa and Zea mays, and seven members in Populus trichocarpa (Jin et al., 2012). In our study, only two members, dlo-miR160a and dlo-miR160d∗, were detected in longan somatic embryos. However, these two members were represented by 12 variants with identical or nearly identical sequences and different read counts. qPCR results further indicated that the different members of the dlo-miR160 family had different expression patterns. A previous study also showed that the multi-copy miR160 family has expression diversification, and may have functional diversity. For example, miR160a regulates embryo development at the very beginning of embryogenesis (Liu et al., 2010b), while miR160c regulates root cap development (Wang et al., 2005), which suggests that the different spatial and temporal expression patterns of the dlo-miR160 family members reflects their functional diversity in longan SE.
The secondary structures of miRNA genes play an integral role in their maturation, regulation and functions (Parizotto et al., 2004; Jiang et al., 2006). miRNA genes have been identified using computational approaches that depend upon the availability of reference or genomic sequences (Megraw et al., 2006; Meng et al., 2009; Branscheid et al., 2011; Han et al., 2014). However, most miRNA genes, especially in non-model plants, have not been investigated, primarily because of a lack of genome-wide datasets. In our study, 5′ and 3′ RACE experiments, together with the data deposited in the GenBank and miRBase databases, were used to obtain the full-length pri-miRNA for miR160. The corresponding pri-miR160a genomic DNA sequence (424 bp, containing one intron) was obtained, which was shorter than the Arabidopsis miR160a gene (2034-bp long; one intron), but longer than the Arabidopsis miR160b gene (378-bp long; no introns) (Szarzynska et al., 2009). This suggests that the pri-miR160 genes from different species and different members vary in size and structure.
The Longan miR160 Promoter is Associated with Hormone Signal Transduction
Many previous studies have focused on elucidating the mechanism of miRNA target gene regulation, but there is limited information on the regulation of miRNA genes themselves. Previous studies have shown that the promoters of most of the known pre-miRNAs share similar characteristics with RNA polymerase II-dependent protein-coding gene promoters (Zhou et al., 2007). The identification of the promoters of miRNA genes is a work in progress; however, most studies have concentrated on A. thaliana (Megraw et al., 2006; Zhou et al., 2007) and O. sativa (Cui et al., 2009; Meng et al., 2009). Here, the pri-miR160 promoter was cloned from longan EC and contained the conserved TATA and CAAT boxes.
The miR160 family has the highest number of identified ARF binding sites (AuxREs) in cassava (Perez-Quintero et al., 2012), G. max (miR160d and miR160f) (Han et al., 2014), and rice (osamiR160f). However, ARF binding sites were not found in the longan miR160 promoter. Previous experiments showed that indole acetic acid (IAA) treatment did not influence the auxin-related rice homologous miRNAs significantly, such as osa-miR160, osa-miR164, and osa-miR167 (Meng et al., 2009). Here, exogenous 2,4-D treatment significantly influenced pri-miR160 expression, suggesting that ARF elements may be present outside of the 960 bp promoter region. Indeed, AuxREs in the 3′ regulatory region of miR160a are required for auxin regulation of miR160a expression in Arabidopsis (Liu et al., 2010b). However, AuxREs were not found in the 3′ regulatory region of pri-miR160.
Other cis-regulatory elements associated with plant hormones, such as ABA (ABRE) and SA (TCA-element), necessary for plant responses to various abiotic stresses (Kruszka et al., 2012), were detected in longan. In Arabidopsis, miR160, miR393 and miR397b were up-regulated, whereas miR169 and miR398 were down-regulated, in response to ABA stress. In another study, negative regulation of ARF10 by miR160 modulated the response to ABA stress during germination (Liu et al., 2007). Our qPCR results showed that ABA up-regulated the expression of miR160, which suggests that miR160 is involved in ABA signaling in longan. Furthermore, pri-miR160 was down-regulated in response to SA treatment, implying that miR160 is also involved in SA signaling in longan. GA3, ET, and MeJA also up-regulated the expression level of pri-miR160, but corresponding cis-regulatory elements were not identified in the 960 bp longan miR160 promoter region. However, gibberellin response factor elements were found in the putative promoter regions of miR160f, miR160g, and miR160h in G. max (Han et al., 2014), and auxin-, MeJA-, ET-, GA3-, and SA-responsive elements were found in the promoter regions of miR160s in cassava (Pinweha et al., 2015). Taken together, these results clearly demonstrated that the pri-miR160 was down-regulated in response to SA (75 μM), but up-regulated by MeJA (50 μM), ET (30 ppm), GA3 (35 μM), or ABA (100 μM), suggesting that miR160s are widely involved in hormone signal transduction in longan. However, it is not clear whether these hormones affect the morphogenesis of somatic embryos; this requires further experimental validation.
The other types of cis-regulatory elements in the upstream region of the miR160 gene in longan were defense and stress responsive, such as the anaerobic-response element (ARE) and HSE, which were also found in the cassava miR160 promoter (Pinweha et al., 2015). Recently, reports showed that miR160 plays a role in the defense and stress responses of plants (Jin et al., 2012; Pinweha et al., 2015). miR160s were highly induced after infection with Pseudomonas syringae pv. tomato (Fahlgren et al., 2007). These results suggest that miR160 might play a role in defense and stress in plants.
In conclusion, these results form the basis for further experiments to identify the function of miR160 in responses to various hormone and environmental stimuli.
eTMs Down-regulated miR160 Cleavage of DlARF10, -16, and -17 mRNAs During Longan SE
Target mimicry, a new regulatory mechanism for miRNA functions, was first studied in Arabidopsis. eTMs for miR160 have been identified in Arabidopsis (Wu et al., 2013), rice (Wu et al., 2013), and P. trichocarpa (Shuai et al., 2014), and are functional target mimics that cause smaller and serrated leaves in transgenic plants (Wu et al., 2013). However, previous studies focused mainly on eTMs derived from intergenic non-coding RNA genes. In our study, four potential eTMs derived from annotated genes were initially identified for miR160a∗ in longan; however, only two eTMs: glucan endo-1,3-beta-glucosidase-like protein 2-like (Unigene64827) and calpain-type cysteine protease DEK1 (Unigene 66891), were detected during longan SE by qPCR. Their expression patterns were inversely proportional to that of the corresponding dlo-miR160a∗, especially at the GE to CE stages. Moreover, their expression was down-regulated while dlo-miR160a∗ was up-regulated by auxin and ABA treatment. These results further proved that Unigene64827 and Unigene 66891 inhibited the expression level of dlo-miR160a∗ and participated in auxin and ABA signal transduction during longan SE. Unigene64827 encodes a homolog of glucan endo-1,3-beta-glucosidase-like protein 2, which is related to cell wall synthesis (Meitzel et al., 2011). Unigene66891 encodes a protein similar to calpain-type cysteine protease DEK1, which is required for epidermal development in the embryo (Johnson et al., 2005). These results suggest that they play a potential role in cell wall synthesis and epidermal development in the embryo by regulating dlo-miR160a∗, especially at the GE to CE stages. This hypothesis requires further experimental validation.
miR160 and its target ARFs are conserved in dicots and monocots (Rhoades et al., 2002). Here, DlARF10, -16, and -17 targeting by dlo-miR160a was confirmed, and their expression levels were mostly the reverse of that of dlo-miR160a. In addition, dlo-miR160a was up-regulated while DlARF16 and -17 were down-regulated by 2,4-D treatment. These results were consistent with earlier reports (Liu et al., 2010b), which suggested that the increased expression of dlo-miR160a induced by 2,4-D treatment down-regulated the expression of ARF16 and -17, further confirming that miR160-ARF16-17 auxin signal transduction operates in longan similarly to Arabidopsis. By contrast, DlARF10 was up-regulated after 2,4-D treatment, suggesting it may not be controlled by dlo-miR160a under auxin treatment.
Auxin response factor proteins can either activate or repress transcription, based on their protein structures and transient expression experiments. ARFs 5–8 and ARF19 contain Gln-Leu-Ser–rich middle regions that may function as transcriptional activators, whereas ARFs 1–4, 9–18, and 20–22 possibly act as repressors, with Pro-Ser-Thr–rich middle regions (Tiwari et al., 2003; Mallory et al., 2005). In our study, DlARF10, -16, and -17 expression was stronger in whole embryos at the EC and ICpEC stages, and gradually decreased during somatic embryo development, suggesting they might function as repressors to establish and maintain auxin signals during embryogenesis (Mallory et al., 2005; Liu et al., 2007, 2010b).
miR160-resistant mutants have been used successfully to characterize the biological functions of ARF10 (Liu et al., 2007), -16 (Wang et al., 2005) and -17 (Mallory et al., 2005), revealing that down-regulation of these genes is essential to maintain the normal developmental programs of the roots, leaves, flowers and siliques. In our study, neither pri-miR160 nor ARF10, -16, or -17 exhibited tissue specificity in 'Sijimi' longan. They all showed high expression in roots and were not detected in mature leaves. By contrast, miR160c expression was low or undetectable in juvenile leaves and became high in older leaves, as assessed by GUS staining of ProMIR160c:GUS plants (Wang et al., 2005). miR160a is expressed in all organs, especially in reproductive organs (Liu et al., 2010b), suggesting that miR160 regulation of ARF10, -16, and -17 signal transduction has an extensive role in 'Sijimi' development. However, there were no remarkable negative correlations among the levels of miR160 and the DlARF10, -16, and -17 mRNAs in the analyzed tissues.
In summary, we performed dlo-miR160 family member identification, primary transcript and promoter cloning; target mimic prediction, miRNA target identification, and qPCR verification. According to our experiments as well as the negative feedback model, we propose a possible "eTM–miR160–target" regulatory loop during longan SE (Figure 13). The eTMs glucan endo-1,3-beta-glucosidase-like protein 2-like (Unigene64827) and calpain-type cysteine protease DEK1 (Unigene 66891) down- regulation of miR160's mediation of ARF10, -16, and -17 cleavage is associated with SE, and responds to 2,4-D and ABA signaling transduction in longan. In addition, the expression of pri-miR160 is controlled by GA3, ET, MeJA, and SA.
FIGURE 13. Negative regulatory relationships among the eTMs, miR160, and its targets involved in SE in D. longan in relation to hormone metabolism. The eTMs Unigene64827 and Unigene66891 belong to the glucan endo-1,3-beta-glucosidase- like protein 2 and calpain-type cysteine protease DEK1 families, respectively. They down-regulate miR160 by mediating cleavage of ARF10, -16, and -17, which are involved in SE in D. longan, and respond to 2,4-D and ABA signal transduction. In addition, the expression of pri-miR160 is controlled by exogenous GA3, ET, MeJA and SA.
YL conceived the study, participated in its design, carried out the experimental work and wrote the manuscript. ZL conceived the study, participated in its design and coordination, and helped to draft the manuscript. QT, LL, RL, MY, DZ, YC, and ZZ prepared the materials. All authors read and approved the final version of the manuscript. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
This work was funded by Research Funds for the Doctoral Program of Higher Education of the Chinese Ministry of Education (20123515120008), the National Natural Science Foundation of China (31201614 and 31272149), the Natural Science Funds for Distinguished Young Scholars in Fujian Province (2015J06004), the Fujian Agriculture and Forestry University Funds for Distinguished Youth Fund (xjq201405), and the Outstanding Youth Fund of the Fujian Provincial Department of Education Teacher Education of Young and Middle-aged Teachers in Science and Technology Research (Science and Technology Class A) (JA14099).
2,4-D, 2,4-dichlorophenoxyacetic acid; ABA, abscisic acid; ARF, auxin response factor; CE, cotyledonary embryos; EC, friable embryogenic callus; ET, ethylene; eTMs, endogenous target mimics; GA3, gibberellic acid; GE, globular embryos; HE, heart- shaped embryos; ICpEC, incomplete compact pro-embryogenic cultures; MeJA, methyl jasmonate; miRNAs, microRNAs; pre-miRNA, precursor miRNA; pri-miRNAs, primary miRNA transcripts; qPCR, real-time quantitative PCR; RLM-RACE, RNA ligase-mediated rapid amplification of cDNA ends; SA, salicylic acid; SE, somatic embryogenesis; TE, torpedo-shaped embryos; UTR, untranslated region.
Bonnet, E., He, Y., Billiau, K., and Van De Peer, Y. (2010). TAPIR, a web server for the prediction of plant microRNA targets, including target mimics. Bioinformatics 26, 1566–1568. doi: 10.1093/bioinformatics/btq233
Branscheid, A., Devers, E. A., May, P., and Krajinski, F. (2011). Distribution pattern of small RNA and degradome reads provides information on miRNA gene structure and regulation. Plant Signal. Behav. 6, 1609–1611. doi: 10.4161/psb.6.10.17305
Carrington, J. C., and Ambros, V. (2003). Role of microRNAs in plant and animal development. Science 301, 336–338. doi: 10.1126/science.1085242
Chung, Y. C., Lin, C. C., Chou, C. C., and Hsu, C. P. (2010). The effect of Longan seed polyphenols on colorectal carcinoma cells. Eur. J. Clin. Invest. 40, 713–721. doi: 10.1111/j.1365-2362.2010.02322.x
Cui, X., Xu, S. M., Mu, D. S., and Yang, Z. M. (2009). Genomic analysis of rice microRNA promoters and clusters. Gene 431, 61–66. doi: 10.1016/j.gene.2008.11.016
Fahlgren, N., Howell, M. D., Kasschau, K. D., Chapman, E. J., Sullivan, C. M., Cumbie, J. S., et al. (2007). High-throughput sequencing of Arabidopsis microRNAs: evidence for frequent birth and death of MIRNA genes. PLoS ONE 2:e219. doi: 10.1371/journal.pone.0000219
Franco-Zorrilla, J. M., Valli, A., Todesco, M., Mateos, I., Puga, M. I., Rubio-Somoza, I., et al. (2007). Target mimicry provides a new mechanism for regulation of microRNA activity. Nat. Genet. 39, 1033–1037. doi: 10.1038/ng2079
Gandikota, M., Birkenbihl, R. P., Hohmann, S., Cardon, G. H., Saedler, H., and Huijser, P. (2007). The miRNA156/157 recognition element in the 3′ UTR of the Arabidopsis SBP box gene SPL3 prevents early flowering by translational inhibition in seedlings. Plant J. 49, 683–693. doi: 10.1111/j.1365-313X.2006.02983.x
Han, Y. Q., Hu, Z., Zheng, D. F., and Gao, Y. M. (2014). Analysis of promoters of microRNAs from a Glycine max degradome library. J. Zhejiang Univ. Sci. B 15, 125–132. doi: 10.1631/jzus.B1300179
Huang, D., Koh, C., Feurtado, J. A., Tsang, E. W., and Cutler, A. J. (2013). MicroRNAs and their putative targets in Brassica napus seed maturation. BMC Genomics 14:140. doi: 10.1186/1471-2164-14-140
Jiang, D., Yin, C., Yu, A., Zhou, X., Liang, W., Yuan, Z., et al. (2006). Duplication and expression analysis of multicopy miRNA gene family members in Arabidopsis and rice. Cell Res. 16, 507–518. doi: 10.1038/sj.cr.7310062
Jin, W., Wu, F., Xiao, L., Liang, G., Zhen, Y., Guo, Z., et al. (2012). Microarray-based analysis of tomato miRNA regulated by Botrytis cinerea. J. Plant Growth Regul. 31, 38–46. doi: 10.1007/s00344-011-9217-9
Johnson, K. L., Degnan, K. A., Ross Walker, J., and Ingram, G. C. (2005). AtDEK1 is essential for specification of embryonic epidermal cell fate. Plant J. 44, 114–127. doi: 10.1111/j.1365-313X.2005.02514.x
Jones-Rhoades, M. W., Bartel, D. P., and Bartel, B. (2006). MicroRNAs and their regulatory roles in plants. Annu. Rev. Plant Biol. 57, 19–53. doi: 10.1146/annurev.arplant.57.032905.105218
Kruszka, K., Pieczynski, M., Windels, D., Bielewicz, D., Jarmolowski, A., Szweykowska-Kulinska, Z., et al. (2012). Role of microRNAs and other sRNAs of plants in their changing environments. J. Plant Physiol. 169, 1664–1672. doi: 10.1016/j.jplph.2012.03.009
Kumar, S., Tamura, K., and Nei, M. (2004). MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief. Bioinform. 5, 150–163. doi: 10.1093/bib/5.2.150
Lai, Z., and Chen, C. (1997). Somatic embryogenesis of high frequency from longan embryogenic calli. J. Fujian. Agric. Univ. 26, 271–276.
Lai, Z. X., He, Y., Chen, Y. T., Cai, Y. Q., Lai, C. C., Lin, Y. L., et al. (2010). Molecular biology and proteomics during somatic embryogenesis in Dimocarpus longan Lour. Acta Hortic. 863, 95–102. doi: 10.17660/ActaHortic.2010.863.10
Lai, Z., and Lin, Y. (2013). Analysis of the global transcriptome of longan (Dimocarpus longan Lour.) embryogenic callus using Illumina paired-end sequencing. BMC Genomics 14:561. doi: 10.1186/1471-2164-14-561
Lee, Y. S., Nakahara, K., Pham, J. W., Kim, K., He, Z., Sontheimer, E. J., et al. (2004). Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117, 69–81. doi: 10.1016/S0092-8674(04)00261-2
Lescot, M., Dehais, P., Thijs, G., Marchal, K., Moreau, Y., Van De Peer, Y., et al. (2002). PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 30, 325–327. doi: 10.1093/nar/30.1.325
Lin, Y. L., and Lai, Z. X. (2010). Reference gene selection for qPCR analysis during somatic embryogenesis in longan tree. Plant Sci. 178, 359–365. doi: 10.1016/j.plantsci.2010.02.005
Lin, Y.-L., and Lai, Z.-X. (2013a). Superoxide dismutase multigene family in longan somatic embryos: a comparison of CuZn-SOD, Fe-SOD, and Mn-SOD gene structure, splicing, phylogeny, and expression. Mol. Breed. 32, 595–615. doi: 10.1007/s11032-013-9892-2
Lin, Y., and Lai, Z. (2013b). Comparative analysis reveals dynamic changes in miRNAs and their targets and expression during somatic embryogenesis in longan (Dimocarpus longan Lour.). PLoS ONE 8:e60337. doi: 10.1371/journal.pone.0060337
Lin, Y. L., and Lai, Z. X. (2013c). Evaluation of suitable reference genes for normalization of microRNA expression by real-time reverse transcription PCR analysis during longan somatic embryogenesis. Plant Physiol. Biochem. 66, 20–25. doi: 10.1016/j.plaphy.2013.02.002
Liu, H., Liu, Y. Z., Zheng, S. Q., Jiang, J. M., Wang, P., and Chen, W. (2010a). Comparative proteomic analysis of longan (Dimocarpus longan Lour.) seed abortion. Planta 231, 847–860. doi: 10.1007/s00425-009-1093-1
Liu, X., Huang, J., Wang, Y., Khanna, K., Xie, Z., Owen, H. A., et al. (2010b). The role of floral organs in carpels, an Arabidopsis loss-of-function mutation in MicroRNA160a, in organogenesis and the mechanism regulating its expression. Plant J. 62, 416–428. doi: 10.1111/j.1365-313X.2010.04164.x
Liu, P. P., Montgomery, T. A., Fahlgren, N., Kasschau, K. D., Nonogaki, H., and Carrington, J. C. (2007). Repression of AUXIN RESPONSE FACTOR10 by microRNA160 is critical for seed germination and post-germination stages. Plant J. 52, 133–146. doi: 10.1111/j.1365-313X.2007.03218.x
Liu, X., Zhang, H., Zhao, Y., Feng, Z., Li, Q., Yang, H. Q., et al. (2013). Auxin controls seed dormancy through stimulation of abscisic acid signaling by inducing ARF-mediated ABI3 activation in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 110, 15485–15490. doi: 10.1073/pnas.1304651110
Llave, C., Xie, Z., Kasschau, K. D., and Carrington, J. C. (2002). Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 297, 2053–2056. doi: 10.1126/science.1076311
Mallory, A. C., Bartel, D. P., and Bartel, B. (2005). MicroRNA-directed regulation of Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper development and modulates expression of early auxin response genes. Plant Cell 17, 1360–1375. doi: 10.1105/tpc.105.031716
Megraw, M., Baev, V., Rusinov, V., Jensen, S. T., Kalantidis, K., and Hatzigeorgiou, A. G. (2006). MicroRNA promoter element discovery in Arabidopsis. RNA 12, 1612–1619. doi: 10.1261/rna.130506
Meitzel, T., Radchuk, R., Nunes-Nesi, A., Fernie, A. R., Link, W., Weschke, W., et al. (2011). Hybrid embryos of Vicia faba develop enhanced sink strength, which is established during early development. Plant J. 65, 517–531. doi: 10.1111/j.1365-313X.2010.04450.x
Meng, Y., Huang, F., Shi, Q., Cao, J., Chen, D., Zhang, J., et al. (2009). Genome-wide survey of rice microRNAs and microRNA-target pairs in the root of a novel auxin-resistant mutant. Planta 230, 883–898. doi: 10.1007/s00425-009-0994-3
Parizotto, E. A., Dunoyer, P., Rahm, N., Himber, C., and Voinnet, O. (2004). In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA. Genes Dev. 18, 2237–2242. doi: 10.1101/gad.307804
Park, S. J., Park, D. H., Kim, D. H., Lee, S., Yoon, B. H., Jung, W. Y., et al. (2010). The memory-enhancing effects of Euphoria longan fruit extract in mice. J. Ethnopharmacol. 128, 160–165. doi: 10.1016/j.jep.2010.01.001
Perez-Quintero, A. L., Quintero, A., Urrego, O., Vanegas, P., and Lopez, C. (2012). Bioinformatic identification of cassava miRNAs differentially expressed in response to infection by Xanthomonas axonopodis pv. manihotis. BMC Plant Biol. 12:29. doi: 10.1186/1471-2229-12-29
Pinweha, N., Asvarak, T., Viboonjun, U., and Narangajavana, J. (2015). Involvement of miR160/miR393 and their targets in cassava responses to anthracnose disease. J. Plant Physiol. 174, 26–35. doi: 10.1016/j.jplph.2014.09.006
Reinhart, B. J., Weinstein, E. G., Rhoades, M. W., Bartel, B., and Bartel, D. P. (2002). MicroRNAs in plants. Genes Dev. 16, 1616–1626. doi: 10.1101/gad.1004402
Rhoades, M. W., Reinhart, B. J., Lim, L. P., Burge, C. B., Bartel, B., and Bartel, D. P. (2002). Prediction of plant microRNA targets. Cell 110, 513–520. doi: 10.1016/S0092-8674(02)00863-2
Saitou, N., and Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425.
Shuai, P., Liang, D., Tang, S., Zhang, Z., Ye, C. Y., Su, Y., et al. (2014). Genome-wide identification and functional prediction of novel and drought- responsive lincRNAs in Populus trichocarpa. J. Exp. Bot. 65, 4975–4983. doi: 10.1093/jxb/eru256
Szarzynska, B., Sobkowiak, L., Pant, B. D., Balazadeh, S., Scheible, W. R., Mueller- Roeber, B., et al. (2009). Gene structures and processing of Arabidopsis thaliana HYL1-dependent pri-miRNAs. Nucleic Acids Res. 37, 3083–3093. doi: 10.1093/nar/gkp189
Tiwari, S. B., Hagen, G., and Guilfoyle, T. (2003). The roles of auxin response factor domains in auxin-responsive transcription. Plant Cell 15, 533–543. doi: 10.1105/tpc.008417
Todesco, M., Rubio-Somoza, I., Paz-Ares, J., and Weigel, D. (2010). A collection of target mimics for comprehensive analysis of microRNA function in Arabidopsis thaliana. PLoS Genet. 6:e1001031. doi: 10.1371/journal.pgen.1001031
Turner, M., Nizampatnam, N. R., Baron, M., Coppin, S., Damodaran, S., Adhikari, S., et al. (2013). Ectopic expression of miR160 results in auxin hypersensitivity, cytokinin hyposensitivity, and inhibition of symbiotic nodule development in soybean. Plant Physiol. 162, 2042–2055. doi: 10.1104/pp.113.220699
Wang, J. W., Wang, L. J., Mao, Y. B., Cai, W. J., Xue, H. W., and Chen, X. Y. (2005). Control of root cap formation by MicroRNA-targeted auxin response factors in Arabidopsis. Plant Cell 17, 2204–2216. doi: 10.1105/tpc.105.033076
Wu, H. J., Wang, Z. M., Wang, M., and Wang, X. J. (2013). Widespread long noncoding RNAs as endogenous target mimics for microRNAs in plants. Plant Physiol. 161, 1875–1884. doi: 10.1104/pp.113.215962
Yan, J., Gu, Y., Jia, X., Kang, W., Pan, S., Tang, X., et al. (2012). Effective small RNA destruction by the expression of a short tandem target mimic in Arabidopsis. Plant Cell 24, 415–427. doi: 10.1105/tpc.111.094144
Yi, Y., Liao, S. T., Zhang, M. W., Shi, J., Zhang, R. F., Deng, Y. Y., et al. (2011). Physicochemical characteristics and immunomodulatory activities of three polysaccharide-protein complexes of longan pulp. Molecules 16, 6148–6164. doi: 10.3390/molecules16076148
Zhang, J., Zhang, S., Han, S., Wu, T., Li, X., Li, W., et al. (2012). Genome-wide identification of microRNAs in larch and stage-specific modulation of 11 conserved microRNAs and their targets during somatic embryogenesis. Planta 236, 647–657. doi: 10.1007/s00425-012-1643-9
Zheng, S. Q., Jiang, F., Gao, H. Y., and Zheng, J. G. (2010). Preliminary observations on the antifatigue effects of longan (Dimocarpus longan Lour.) seed polysaccharides. Phytother. Res. 24, 622–624. doi: 10.1002/ptr.2963
Zhou, X., Ruan, J., Wang, G., and Zhang, W. (2007). Characterization and identification of microRNA core promoters in four model species. PLoS Comput. Biol. 3:e37. doi: 10.1371/journal.pcbi.0030037
Keywords: Dimocarpus longan, somatic embryogenesis, microRNA160, endogenous target mimics, auxin response factors, gene regulation
Citation: Lin Y, Lai Z, Tian Q, Lin L, Lai R, Yang M, Zhang D, Chen Y and Zhang Z (2015) Endogenous target mimics down-regulate miR160 mediation of ARF10, -16, and -17 cleavage during somatic embryogenesis in Dimocarpus longan Lour. Front. Plant Sci. 6:956. doi: 10.3389/fpls.2015.00956
Received: 05 August 2015; Accepted: 20 October 2015;
Published: 05 November 2015.
Agnieszka Ludwików, Adam Mickiewicz University, Poland
Biswapriya Biswavas Misra, University of Florida, USA
Taras P. Pasternak, University of Freiburg, Germany
Copyright © 2015 Lin, Lai, Tian, Lin, Lai, Yang, Zhang, Chen and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Zhongxiong Lai, laizx01@163.com


ANAHUAC, Texas (AP) — A southeast Texas sheriff said Sunday that two bodies have been recovered at the site where a Boeing 767 cargo plane crashed into a coastal bay. All three people aboard the Flight 3591 died, according to the plane's owner.
Crews continued to search for the third body at Trinity Bay, about 35 miles (55 kilometers) east of Houston, Chambers County Sheriff Brian Hawthorne said at a Sunday afternoon news conference with officials from the National Transportation Safety Board and the FBI.
The plane's owner, Atlas Air, confirmed the deaths in a statement on Sunday, and said its "primary focus is working to provide the families of those affected with care and support." Atlas was operating the flight for Amazon when it crashed Saturday afternoon near the small town of Anahuac.
Mesa Airlines said in a statement late Sunday that one of its employees, Sean Archuleta, was one the plane. Archuleta had been a pilot for Mesa since 2013.
The other two people have not yet been identified.
NTSB Chairman Robert Sumwalt said security video from a jail about a mile away from the crash site showed the plane heading toward the ground nose first. Sumwalt added that air traffic controllers reported rain in the area, and that the plane did not send out a distress call before the wreck.
Civilian volunteers in small boats helped search part of the bay, but Hawthorne told The Associated Press before the news conference that the volunteers will no longer be used.
Jason Campbell was among the civilian boaters who checked the debris on Saturday. What the boaters found was grim.
Sumwalt said that finding the flight recorders remains a high priority for searchers.
The jumbo jet had departed from Miami and was likely moments from landing at Bush Intercontinental Airport in Houston when it crashed. FBI Special Agent in Charge Perrye Turner asked that anyone with debris on their property contact authorities.


A talk about 'The very odd invasion of Scarborough 1557' will be given by David Rumbelow to the Pocklington and District Local History Group next Thursday (18 February).
The talk will be in the old courthouse, George Street, Pocklington at 7.30pm.
All are welcome and there will be an admission charge of £2.


text:
Department of Science and Environment
Activities (1807)
2791 Journal article
201 Dataset
197 Ph.D. thesis
190 Poster
133 Conference abstract for conference
63 Compendium/lecture notes
37 Anthology
37 Conference abstract in proceedings
34 Conference abstract in journal
17 Other contribution
16 Literature review
3 Contribution to newspaper - Review
1 Memorandum contribution
1 Question & Answer/hearing
Conference abstract in proceedings
Statistics as a tool for decision-making: Two countries and one pattern
Elicer, R. & Franco Neto, V., 2018, Proceedings of the First PME Regional Conference: South America 2018. Gómez, D. M. (ed.). Rancagua, Chile: PME, p. 150 1 p.
Land valuation and marginalization processes in cultural landscapes - a comparative study of valuation systems related to natural and semi-natural areas
Christensen, A. A., Svenningsen, S. R. & Brandt, J., 2012, PECSRL The permanent european conference for the study of the rural landscape, 25th session, 2012: Abstracts of presentations. p. 21 1 p.
Research output: Chapter in Book/Report/Conference proceeding › Conference abstract in proceedings › Research
Agroparks - The European Landscape Convention and a European way to regional sustainable landscape development through land use integration
Christensen, A. A., Svenningsen, S. R. & Brandt, J., Aug 2011, The 8th World Congress of the International Association for Landscape Ecology (IALE). Proceedings: Landscape ecology for sustainable environment and culture. Beijing, China, Vol. 1. p. 91-92 2 p.
Parks & Benefits: Landscape Sustainability in and around Nature Parks
Brandt, J., Christensen, A. A., Holmes, E. & Svenningsen, S. R., 18 Aug 2011, The 8th World Congress of the International Association for Landscape Ecology. Proceedings: Landscape ecology for sustainable environment and culture. Beijing, Kina, Vol. 1. p. 49-50 2 p.
Quantification of landscape multifunctionality based on farm functionality indices
Andersen, P. S., Vejre, H., Dalgaard, T. & Brandt, J., 18 Aug 2011, The 8th World Congress of the International Association for Landscape Ecology (IALE). Proceedings: Landscape ecology for sustainable environment and culture. . Beijing, Kina, Vol. 1. p. 12-13 2 p.
Analysing the functions of mathematical modelling in science as a basis for developing modelling competency in science education
Kjeldsen, T. H. & Blomhøj, M., 2009, ICTMA 14: Abstracts. p. 19 1 p.
Challenges for weighing evidence in new European ecological legislation
Calow, P. & Forbes, V. E., 2009, Abstract Book: SETAC North America 30th Annual Meeting. Society of Environmental Toxicology and Chemistry, p. 105 1 p.
Degradation chemistry of N719 and Z-907 dyes at elevated temperatures
Lund, T., Nguyen, H. T. & Phuong, N. T., 2009, Catalogue of abstracts. Tallin University of Technology, p. 35 1 p.
Lund, T., Nguyen, H. T. & Phuong, N. T., 2009, HOPV09 - Hybrid and Organic Photovoltaics Conference: Book of Abstracts. p. 34 1 p.
Delayed hatching eggs in the copepod Acartia tonsa
Drillet, G., 2009, A Cruise Through Nice Waters. ASLO Aquatic Sciences Meeting 2009: Meeting abstracts. p. 70 1 p.
Demographic importance of toxicant-sensitive life-cycle traits - insights from a matrix population model
Forbes, V. E., Palmqvist, A., Calow, P. & Olsen, M., 2009, Abstract book. Society of Environmental Toxicology and Chemistry
Didactical contract: An analytical concept to facilitate successful implementation of open-ended physics labs
Jacobsen, L. B., Friis Johannsen, B., Rump, C. Ø. & Jensen, J. H., 2009, Ikke angivet: ESERA 2009 Conference Book. European Science Education Research Association, p. 89-89 1 p.
Integrating population modeling into ecological risk assessment.
Forbes, V. E., Palmqvist, A. & Salvito, D., 2009, Abstract Book: SETAC North America 30th Annual Meeting. Society of Environmental Toxicology and Chemistry, p. 81 1 p.
Investigating the Need for Teachers and Students to understand the Purpose of Experimental Work in Physics
Jacobsen, L. B., 2008, The 9th Nordic Research Symposium on Science Education: Symposium program and abstracts. Stefánsson, K. K. (ed.). Stofnun Árna Magnúrssonar á Íslandi, p. 87 1 p.
Assessing ecorelevance of emerging chemicals in sediments
Forbes, V. E., Selck, H. & Salvito, D., 2007, Abstract Book: SETAC North America 28th Annual Meeting. Society of Environmental Toxicology and Chemistry, p. 142 1 p.
Cadmium induced oxidative stress in kidney epithelia cells: involvement of mitochondrial reactive oxygen species (ROS) production
Bjerregaard, H. F., 2007, Proceedings from The 25th Workshop of the Scandinavian Society for Cell Toxicology in Salzau, Germany, 19 - 22 September.. p. 24 1 p.
Discrepancies in scale between stratified samling based landscape monitoring and CORINE land cover registration
Levin, G., Brandt, J., Olsen, M. & Holmes, E., 2007, Proceedings from the 7th World Congress of IALE, Wageningen.
Heilesen, S. & Josephsen, J., 2007, CAL'07 Development, Disruption & Debate - D3, Abstract Book. Tangney, B. (ed.). Pergamon Press, p. O80 1 p.
Interface between urban and rural
Brandt, J., 2007, Interfaces in the study of rural landscapes: Proceedings from the 22th Permanent European Conference for the Study of Rural Landscapes (PECSRL), Berlin and Brandenburg, 3-9 September 2006. Born, K. M., Höchtl, F. & Pliniger, T. (eds.). Freie Universität Berlin
Method testing for habitat requirement sampling in small lowland Danish streams
Conallin, J., Pedersen, S., Jensen, J. K. & Boegh, E., 2007, Ikke angivet. Danish Institute for Fisheries Research, Department of Inland Fisheries
The development of habitat suitability curves for use in Brown Trout (Salmo Trutta) habitat modelling in small lowland streams under Water Framework Directive conditions
Conallin, J., Pedersen, S., Jensen, J. K., Jowett, I. & Boegh, E., 2007, ECEM '07 Conference proceedings, Trieste, Italy, November 27-30, 2007: Challenges for ecological modelling in a changing world: Global Changes, Sustainability and Ecosystem Based Management. Istituto Nazionale di Oceanografia e di Geofisica Sperimentale, p. 124-125 2 p.
Water balance evaluation using remote sensing driven land surface modeling and spatially distributed modeling
Boegh, E., Christensen, B. S. B. & Troldborg, L., 2007, Earth: Our Changing Planet.: Proceedings of IUGG XXIV General Assembly. Umbria Scientific Meeting Association, p. 4219-4220 1 p.
A fast scheme for mapping leaf chlorophyll and leaf area index using inverse canopy reflectance modeling and SPOT reflectance data
Houborg, R., Bøgh, E., Schelde, K. & Thomsen, A., 2006, Eos Trans. AGU. American Geophysical Union, Vol. 87(52). 1 p.
Cadmiun induced cell death in cultured distal kidney epithelial: Discussion of mechanisms and end points.
Bjerregaard, H. F., 2006, Proceedings from the 24th SSCT Annual Workshop on In Vitro Toxicology, Grinda (Stockholm), Sweden. p. 1-15 1 p.
Comparative specific growth and carbon demand for meroplankters and holoplankters in a highly productive Danish estuary, Isefjord
Hansen, B. W. & Lech Fotel, F., 2006, Larval 2006: 7th Larval Biology Symposium, Coos Bay, Oregon August 27 - September 1, 2006. Oregon Institute of Marine Biology, University of Oregon, 1 p.
Effects of flood created decreases in salinity on the fitness of the colonial bryozoan Bugula neritina
Hansen, B. W., 2006, International Conference on Recent Advances in Marine Antifouling Technology: 6 - 8 November, 2006. 1 p.
Formation of the N-Iodopyridinium Cation in an Alkane Environment: Mid-Infrared Linear Dichroism Investigation of the Pyridine-Iodine System in Stretched Polyethylene
Spanget-Larsen, J., 2006, XVIII International Conference on Physical Organic Chemistry: ICPOC-18, August 20-25, 2006. Cyranski, M. K., Wozniak, K. & Krygowski, T. M. (eds.). International Union of Pure and Applied Chemistry, p. 42-43 2 p.
Intensive production of calanoide copepods and possible preservation of COPEPOD eggs for fish farming: Presentation of overall results from the FP5-craft-project, POCEFF
Engell, KS., Drillet, G., Hansen, B. W., Echabe, JA., Evjemo, JO., Fintikaki, M., Holmstrup, M., Overrein, I., Ramløv, H., Overgaard, J., Siampas, T., Svendsen, AK., Thinggaard, A., Tsovenis, I. & Verriopoulis, G., 2006, AQUA 2006 - Meeting Abstract. p. 1060
Micellization and Dynamics of a Block Copolymer
Hvidt, S., 2006, Structure and Dynamics of Self-organized Macromolecular Systems. Institute of Macromolecular Chemistry, Prague, p. 47-47 1 p.
Multi-scale water balance evaluation using remote sensing and agro/eco-hydrological modelling
Bøgh, E., Poulsen, R. N., Butts, M., Dellwik, E. & Søgaard, H., 2006, EOS : Transactions, AGU. American Geophysical Union, Vol. 87(52). 1 p.
Perspectives in Calanoid Copepods as live feed for marine fish larvae
Hansen, B. W., 2006, 7th Asia Pacific Marine Biotechnology Conference: Taj Residency, Kochi, Kerala, India, 2 - 5 November 2006. 1 p.
Remote sensing estimation of atmospheric and land surface parameters for evapotranspiration calculation
Boegh, E., Houborg, R. & Søgaard, H., 2006, Geophysical Research Abstracts. 8(10199) ed. European Geosciences Union, 1 p.
Alumped parameter model predicting cerebral autoregulation and autonomic regulation in young people
Olufsen, M. S., Tran, H. T., Ottesen, J. T., Ellwein, L., Lipsitz, L. A. & Novak, V., 2005, SIAM Conference on Control and its Application. Society for Industrial and Applied Mathematics, 1 p.
Mathematicians doing physics: Mark Kac's Work on the Modeling of Phase Transitions
Niss, M., 2005, Mathematics in the physical sciences, 1650-2000. Mathematisches Forschungsinstitut Oberwolfach, Vol. 56.
Online simulation of classical inorganic analysis - interactive, self instructive simulations give more lab-time
Josephsen, J., 2005, European Variety in Chemistry Education: book of abstracts. Ciesla, P., Maciejowska, I. & Sadowska, A. (eds.). Faculty of Chemistry, Jagiellonian University, p. 62-64
The concept of landscape multifunctionality: Land use as interface between multifunctional agriculture and multifunctional landscapes
Vejre, H., Andersen, E., Brandt, J., Busck, A., Dalgaard, T., Frederiksen, P., Hasler, B., Huusum, H., Høyring, K., Kristensen, L., Kristensen, S., Noe, E., Præstholm, S., Schou, J. & Tybirk, K., May 2005, Abstracts of International Conference on Multifunctional Land use 26-28 May 2005: Meeting Future Demands for Landscape Goods and Services. Mander, Ü. & Meier, K. (eds.). Tartu, Estonia: Tartu University Press, p. 46 1 p.
Modeling autonomic and autoregulation of blood flow during posture change from sitting to standing
Olusen, M. S., Tran, H. T. & Ottesen, J. T., 2004, SIAM conference on life sciences. Society for Industrial and Applied Mathematics, 1 p.

redpajama_set_name: RedPajamaCommonCrawl


We perform every last Saturday of the month at The Esquire Theatre and perform many travel shows too.
Denver's award winning and world record holding shadow cast!
Shadow casting is when a group of costumed performers shadow the screening of a movie. During the show YOU will be expected to yell, dance, and throw things. This is an audience participation movie!
Formed in 2000, Colorado's Elusive Ingredient is a nationally recognized cast with many accomplishments such as holding the world record for the largest Rocky showing of over 8,081 tickets sold! Colorado's Elusive Ingredient performs monthly at The Esquire Theatre and performs many travel shows throughout the year. Rocky Horror is the longest-running release in film history and has a large cult following drawing in crowds weekly throughout the world.
See the links below for pictures from some of our most recent shows.
To see pictures from other past shows see the Past Shows section of our website.


Although El Hierro lacks the theme parks and myriad organised activities of the larger Canary Islands, there's still plenty of good, clean, healthy outdoors fun for children here. Several of the natural pools dotted around the island offer safe swimming, including La Maceta, La Caleta and wildly beautiful Charco Azul. Older children will enjoy the snorkelling and diving around La Restinga and everyone likes learning about giant lizards at La Frontera's Ecomuseo de Guinea & Lagartorio. Up on the inland plateau there are plenty of chances for youngsters to burn off excess energy on fairly gentle hiking trails and at fantastic lookout points.
Much of the island's accommodation is family-friendly (including self-catering apartments and independent casas rurales) and there's much less of a late-night social and dining scene here than elsewhere across Spain, which means kids can be off to bed early. Pavements are generally in good condition, though streets are often steep and few restaurants have baby-change facilities.


James is an entrepreneur who has over 20 years of experience in the construction field, along with extensive experience in property management, project management, development and land assembly. His breadth of experience is vast, both in commercial and residential environments. As a professional property inspector and real estate investor, his insights within the real estate industry are invaluable.
James plays a key role in several aspects of the development process, including working closely with our architects, engineering and construction partners, along with land acquisition, development and financing components within the company.
Dezso Farkas holds two law degrees from Osgoode Hall Law School. In addition to having researched and published in the area of corporate governance, Dezso had the privileged of working with the Institute for Global Law and Policy at Harvard Law from 2014-2016. Dezso has been a real-estate entrepreneur for a number of years and has acted on several complex transactions for clients, as a business lawyer.
As head of Foresite Developments legal team, Dezso plays a key role in several aspects of the development process, including working closely with architects, engineering and construction partners, along with land acquisition, development and financing components within the company.


text:
Senate OKs Peacetime Draft,
But Bars War-Profits Ceiling
Bi-Partisan Gang Kills Civil Rights Amendments
From The Militant, Vol. 12 No. 25, 21 June 1948, p. 1.
JUNE 16 – For the second time in American history the U.S. Senate has voted to establish a conscription system in peacetime. This happened first in 1940, when Washington was making active preparations to enter World War II. The 1948 conscription system is a similar proposition: a step toward war and the further militarization of the country.
Under the terms of the bill adopted by the Senate, all men between the ages of 18 and 25 will have to register for the draft, with the 18 year olds having the "privilege" of volunteering for one year's service, and the others subject to a two year draft. For this, the young men and their families can thank both the Republican and Democratic servants of Wall Street, who passed the bill by a vote of 78 to 10.
This bi-partisan coalition 19 ready to use the youth as cannon-fodder, but they take an entirely different attitude to the employers.
When William Langer (R.-N.D.) proposed the payment of "fair and just compensation, but not to exceed 10% on invested capital" for articles, materials, plants and other facilities received or used by the armed forces, he was overwhelmingly defeated on a voice vote.
"Abnormal" Profits
The second amendment, offered by W. Lee O'Daniel (D-Tex.), proposed the suspension of the draft until Congress passed a law putting a 100% tax on "abnormal" profits from contracts between employers and the armed services. This was beaten down by a vote of 81 to 8. Big profits never seem abnormal to Big Business and its political hired help.
The over-all reactionary nature of the bill was underlined by a number of other actions taken by the Senate:
It defeated by a standing vote a proposal to put the general management of the draft into the hands of civilians, rather than the brass hats. One section of the bill provides for setting up an American Foreign Legion, to be composed of 25,000 aliens enlisted here and abroad, for occupational and other services abroad. The use of mercenaries in legions of this kind has long been a hallmark of imperialist policy.
The defeat of the Langer civil rights amendments (except the relatively harmless one against the payment of poll taxes by servicemen) was a demonstrative stub in the back to the Negro struggle for equality in the armed forces. Spokesmen of both parties claimed that these amendments should be considered separately from the draft bill itself. But such claims dripped with hypocrisy because neither party made any effort to consider such proposals separately, or any other way.
The House of Representatives is now considering a conscription bill similar to the one passed by the Senate, although it has tentatively reduced the draft period to one year. Its passage in the House would increase the armed forces from about ½ million to 2 million persons, and the organized reserves from about 1 million to 1½ million.
The duration of the draft measure, as set in the Senate bill, is two years. But actually, it is intended as the beginning of a permanent conscription system, and will surely be extended at the end of the two years if the capitalist parties remain in control. That has always happened in all countries resorting to peacetime conscription.


Good Size 4 Bedroom Family Cottage Situated On Good Size Lot And Steps Away From Water Access On Canal Lake Offering Excellent Boating Swimming And Fishing. Some Tlc Required. Kitchen Offers Walkout To Covered Deck.


text:
Das Rathaus von Kaiserslautern wurde 1968 eingeweiht.

Geschichte
Der Entwurf des Architekten Roland Ostertag aus Leonberg war im Jahr 1960 aus einem Architektenwettbewerb für das Rathaus am Paradeplatz in Mannheim als Sieger hervorgegangen, wegen Finanzierungsschwierigkeiten wurden aber nur die Fundamente fertiggestellt (siehe Mannheimer Stadthaus). Mit kleinen Veränderungen wurde dieser Entwurf dann in Kaiserslautern realisiert. Der Grundstein für das Rathaus wurde im Juli 1964 gelegt; nach vierjähriger Bauzeit wurde es am 29. November 1968 eingeweiht.

Das Rathaus von Kaiserslautern gilt heute als Hauptwerk des 2018 gestorbenen Architekten Ostertag.

Merkmale
Das Gebäude ist mit 22 oberirdischen Stockwerken 84 Meter hoch. Im 22. Stock befindet sich die Dachterrasse und im 21. Stockwerk eine Lounge mit Bar. Die drei obersten Stockwerke werden nur für Lagerzwecke und Betriebstechnik genutzt. Vier Aufzüge im Gebäudekern erschließen alle Stockwerke.

Institutionen
In diesem Gebäude ist ein Großteil der Stadtverwaltung Kaiserslauterns untergebracht.

Außerdem beherbergt dieses Gebäude die Notverwaltung des Katastrophenschutzes, welche sonst bei der Feuerwehr Kaiserslautern liegt.

Weblinks

 Artikel zum 50. Jubiläum der Einweihung auf kaiserslautern.de

Einzelnachweise

Bauwerk in Kaiserslautern
Kaiserslautern
Bauwerk des Brutalismus in Rheinland-Pfalz
Erbaut in den 1960er Jahren
Hochhaus in Rheinland-Pfalz

redpajama_set_name: RedPajamaWikipedia


text:
Tea Partiers take back country
Home » Latest News » Tea Partiers take back country
Posted By Brittany Montgomery on Apr 15, 2010
Conservative republicans met in Salado, Texas on Thursday evening as part of the Taxed Enough Already organization, hoping to educate the public on political candidates and encourage them to be active in the next election.
Judy Brady, organizer of the Tea Party, estimated 1,000 people attended the rally. "We are tired of our government representatives ignoring what we say," Brady said, "and not following the constitution."
Brady hoped to bring people together who are tired of the changes the government is making and are worried about the future. "We want the same opportunities for our children and grandchildren that we had," she said.
Many protestors held their own signs while listening to speakers. Photo by Matthew Peterson
Retired Lt. Gen. Dave Palmer was also one of the speakers at the Salado Tea Party. Addressing the crowd he said, "When we say 'Take back our country' that's exactly what we mean"
Rayford Brown, a retired Lt. Col. from Temple, Texas said, "We hope to motivate people to become involved in government because if they don't apathy will become tyranny."
Brown was one of the headlining speakers at the event, which was held at Wildfire Ranch Arena. He said, "The job of the government is the common defense and welfare of the U.S. and there is no restrictions on the states. The federal government's job is to protect the states as a whole."
Brown spoke to the crowd about constitution patriotism and focused on the changes that have taken place in the words of the Constitution over the past century. "The progressive movement started about 100 years ago and has been intermittently changing our government from a republic to a tyranny by the Supreme Court rewriting laws and changing our Constitution."
Brown's desire is to see the federal government work for the welfare of the states within the boundaries of the laws and regulations already present. He said, "Our government has made a lot of promises they can't keep. We're trying to restore the constitutional government under its original intent."
Author: Brittany Montgomery


Begin your day with some culture & history, the tour will take you through the streets of Oxford abundant with University buildings and famous architecture that inspired artists and poets through the ages.
As you ride through the leafy suburbs of the residential part of the city you are heading towards the atmospheric Cherwell Boathouse and riverside restaurant. Here you will enjoy a romantic boat ride along the Thames and experience parts of the University and Town only accessible from the River. There is exquisite riverside dining available at the boathouse restaurant and even a bottle of champagne for the boat if your heart so desires.
We leave the city along one of the tranquil waterways and ambles through villages outside of the city, some of which date back to Saxon times. An afternoon drink or snack can be enjoyed at one of our carefully selected country Inns or whilst visiting the remains of a 1st century Roman Villa.
The final part of the tour takes you through unblemished countryside towards the charming English village of Minster Lovell, where you will find your final destination The Old Swan and Minster Mill. "At the heart of the Old Minster village and with a history going back almost 600 years, The Old Swan and Minster Mill couldn't be more quintessentially English. The perfect spot to escape the city and explore the Cotswolds. Or just relax, spa and dine".
Additional nights and spa treatments are also available.


OSA values respecting individual dignity, reaching pick point of their career development and the growth of self-actualization. It is not just about teaching students to be good for their academic success rather nurturing them to be their best.
OSA prepares EU students for the leadership role which is accomplished through effectiveness of OSA's programs and services and using reliable information to support responsible decision-making. OSA provides opportunities to develop the leadership competencies that will allow students to create meaningful and sustainable change in their respective societies.
OSA's strong community partnerships and deep involvements actively engage students in the activity and promote social justice and civic engagement. OSA offers a safe, open and welcoming community that nurtures healthy personal development and a sense of belonging to everyone in the community through respect.
OSA values charting the way towards excellence for all through pursuing diversity in culture, equity in education and integration of being respectful, honest and ethical.


text:
PROTECT YOURSELF FROM POISONING
One in five carbon monoxide detectors 'dodgy' – how to spot if yours is a fake
The colourless, odourless and tasteless gas kills 50 people every year
Amanda Devlin
MILLIONS of lives are being put at risk by faulty carbon monoxide detectors as it's revealed one in five are "dodgy".
Shockingly, 50 people die from the "silent killer" - a colourless, odourless and tasteless gas - every year, with four people being admitted to hospital every day.
Three out of 16 carbon monoxide alarms tested by Which? failed EU regulations
An investigation by Which? found that three out of 16 detectors failed lab testing based on EU safety standards.
Detectors made by ATZ Saviour, Binwo and Mudder failed to go off 26 times during 72 carbon monoxide tests, the consumer group said.
Which? is now warning consumers not to buy alarms made by these brands.
SILENT KILLER IN OUR HOMES
Newlywed's tragic death sparks warning over two fifths of Brits not having carbon monoxide alarm
FLOOD DEATH TRAGEDY
Boy, 7, died from carbon monoxide poisoning caused by water pump rented by parents during flooding
'I COULD HAVE STOPPED HIM'
Read the disturbing texts that reveal girlfriend encouraged suicidal lad, 18, to kill himself
2 DIE FROM E.COLI LINKED TO SALAD
Major health fears over bags containing Mediterranean rocket
Were you a chubby teenager? Then you'll need to hear this urgent health warning
Since the investigation, Amazon and eBay have removed the three defective alarms from sale.
The other 13 alarms from established brands passed all of the gas tests and also carry the BSI Kitemark.
Which? editor Richard Headland said: "It's shocking to uncover the sheer volume of carbon monoxide detectors out there that simply don't work.
"Consumers need to be aware that there are dodgy detectors on the market and should only buy products bearing the BSI Kitemark".
What is carbon monoxide poisoning?
CO poisoning is extremely dangerous and is often dubbed the silent killer.
Potentially deadly carbon monoxide is created when a fuel isn't burning correctly.
The gas is usually emitted from faulty gas boilers, fires and cookers and has no colour, taste or smell.
Symptoms of carbon monoxide poisoning include headaches, dizziness, weakness and chest pain.
If exposed to carbon monoxide for longer, symptoms will worsen to confusion, memory loss, co-ordination problems, personality changes or unusual behaviour, tachycardia (a heart rate of more than 100 beats a minute), chest pain caused by angina or a heart attack, seizures or muscle spasms and loss of consciousness.
Low exposures over time, which goes undetected, can cause permanent lung damage, heart damage, brain damage or death.
Most victims die in their sleep from a strong leak. Sometimes it can only take a few minutes.
Family pets are often the first to die before babies, small children or the elderly.
Badly installed or poorly serviced appliances can cause gas leaks, fires, explosions and carbon monoxide poisoning.
In the UK, carbon monoxide poisoning in UK homes kill 50 people ever year, according to the Department of Health.
Ten years ago during a family holiday in Corfu, siblings Bobby, six, and Christi Shepherd, seven, died from carbon monoxide poisoning after a faulty boiler leaked deadly gas into their bedroom.
It's shocking to uncover the sheer volume of carbon monoxide detectors out there that simply don't work.
Richard HeadlandWhich? editor
Carbon monoxide is colourless, odourless and tasteless making it a silent killer
Back in Britain there are as many as 4,000 medical visits, with four people admitted to hospital for related injuries each day and one in five requiring treatment to their lungs, CO-Gas Safety says.
Graham McKay, Global Head of Gas & Electrical Products at BSI said: "The UK is one of the safest countries in the world when it comes to gas related incidents and much of this is due to the rigorous industry standards we have in place – mostly developed and published by BSI.
How do you know if you've got a faulty carbon monoxide detector?
The most common carbon monoxide detector problems include the following:
If the alarm goes off it is safe to assume carbon monoxide has been detected, but in some cases it could be a fault in the alarm. In this case the alarm fault indicator will show a combination of flashing LEDS, or an amber LED flashing.
If this happens, call a technician to fix the problem or have it replaced.
If the alarm is softly beeping it is likely to be a warning about low battery. It can be heard every 30 seconds.
You should always replace your batteries immediately your carbon monoxide detector sounds the low battery warning.
The third most common problem is substandard detectors. Alarms measuring the presence of carbon monoxide must have the correct calibrations so that it can sense low levels of carbon monoxide concentration. Some detectors in the market only detect carbon monoxide concentration that is significantly above safe levels.
Carbon monoxide detectors should be replaced after every five to seven years.
"However, with one in 11 boilers found to be unsafe in 2015 and only half of UK homes fitted with carbon monoxide alarms, we strongly encourage the British public to address the threat of this silent killer by purchasing alarms.
"Importantly though, when shopping for appliances people should be looking out for trust marks such as the Kitemark™ or the Gas Safe registration to make sure the gas products they buy, as this indicates that those products have been tested to the highest possible standard."
8 signs YOU'VE got a carbon monoxide leak
1. LOOK for stains, soot or discolouration on or around your boiler, fire or water heater. Flames of a lazy yellow or orange colour on your gas hob, rather than being a crisp blue;
2. INCREASED condensation inside windows. Make sure the air vents in your doors, walls and windows clear.
3. MAKE SURE there is plenty of ventilation and no blockages in your chimney or flue.
4. CHECK the pilot light on your boiler. They normally burn blue but if the flame is yellow or orange, you may have a carbon monoxide leak.
5. BE VIGILANT for warning signs. Be cautious if there is a stale, stuffy smell in a clean home or even a burning smell. It may not be from carbon monoxide itself, but from other toxic gases being emitted by malfunctioning equipment.
6. SYMPTOMS of carbon monoxide poisoning include headaches, dizziness, weakness and chest pain. If you suspect CO poisoning get fresh air immediately, open doors and windows, turn off gas appliances and leave the house.
7. SAFETY CHECKS during a boiler service must be carried out by a Gas Safe Register engineer, or OFTEC registered for oil-fired boilers to alert you to the presence of the poisonous gas in your home before it's too late.
8. DETECTORS save lives so they should be installed on every floor of your home (especially in all of the bedrooms) and in every room with a fuel-burning appliance.
Cheap detectors only change colour when it detects carbon monoxide so buy an audible alarm to wake you up during the night.


Terminal doors open at approximately 3:30 a.m. daily, and close at approximately 12:00 midnight.
The Quad City International Airport, located in Moline, IL, serves travelers from throughout Eastern Iowa and Western Illinois. Four major airlines offer nonstop daily service to 10 cities- most of which are very active connecting hubs, with domestic and international connection possibilities. Airlines include American, Allegiant, Delta Air Lines, and United Express. (Allegiant flights not daily.) Cities served include Atlanta, Chicago O'Hare, Dallas-Ft. Worth, Denver, Detroit, Ft. Myers (via Punta Gorda, FL) Las Vegas, Minneapolis-St. Paul, Phoenix-Mesa, Orlando-Sanford and St. Petersburg/Clearwater Beach. *Ft. Myers seasonal.
Follow 67S into Bettendorf. Enter I-74E ramp. Follow I-74 to intersection of Hwy.6 and into airport. Or, follow Hwy 30 East to DeWitt, Hwy 61 S to I-80E, to I-74E. Follow to intersect. of Hwy 6.


Pale green glass portrait flask with curved edges and a short neck. On one side is a bust of man in uniform, nearly full face (identified as Lafayette). On the other side is an eagle with a shield that has eight vertical bars. There is a thunderbolt and an olive branch in the eagle's talons.


Brittin - Name Meaning, What does Brittin mean?
Brittin as a name for girls is of Latin origin, and the meaning of the name Brittin is "from Brittany or Britain". Brittin is an alternate form of Brittany (Latin).
Recommended sound-alike names are Baibin, Battina, Beattie, Beitris, Betti, Bettie▼, Bettina, Bettine, Bithia, Bitta, Brandin, Brettany, Briann, Bridgid, Bridgit, Bridi, Bridie, Brieann, Brieon, Briet, Brieta, Brietta, Briette, Brighid, Brigid, Brigit, Brijit, Brilie (see Briley), Brinn, Brioni, Briseis, Brisia, Bristol▲, Brita, Britania, Brites, Brittanya, Bronwin, Caitlin▼, Caitrin, Christin▼, Cristin, Crystin, Kaitlin▼, Kaitrin, Khristin, Kristain, Kristin▼, Krystin, Tristin and Trystin. These names tend to be more commonly used than Brittin.


If you're working on New Year's Eve and want to celebrate New Year's Day instead, we get it! In the hospitality industry, holidays are big money-making days, and you usually don't get them off! So celebrate a day late and grab a bite to eat and a round of drinks with your friends to ring in the new year at any of these restaurants open on New Year's Day in St. Petersburg and Clearwater.


From information collected in a national survey of optometrists conducted by the National Center for Health Statistics, statistics are presented describing features of optometric practice in the United States in 1968. These features include the form and composition of practice arrangements, primary and secondary activities of the practitioners, the range of services that practitioners provided and the numbers and types of supplementary personnel that the optometrist used to assist him in his practice.
State legislation for physician's assistants. A review and analysis.
5-year followup of the effect on optometrists of continuing education about hypertension.
The participation of optometrists in New York City's Medicaid program.
Designation of health manpower shortage areas for use by public health service programs.