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Original Research ARTICLE
Overexpression of PvGF14c from Phyllostachys violascens Delays Flowering Time in Transgenic Arabidopsis
- 1The State Key Laboratory of Subtropical Silviculture, Zhejiang A & F University, Hangzhou, China
- 2School of Geography and Remote Sensing, Nanjing University of Information Science and Technology, Nanjing, China
- 3Institute of Biotechnology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China
- 4Zhejiang Provincial Collaborative Innovation Center for Bamboo Resources and High-efficiency Utilization, Zhejiang A & F University, Hangzhou, China
14-3-3 Proteins are a family of highly conserved regulatory molecules expressed in all eukaryotic cells and regulate a diverse set of biological responses in plants. However, their functions in flowering of Phyllostachys violascens are poorly understood. In this study, four non-𝜀 Pv14-3-3 genes from P. violascens were identified and named PvGF14b, PvGF14c, PvGF14e, and PvGF14f. qRT-PCR analyses revealed that PvGF14b and PvGF14e exhibited widely expressed in all tested bamboo tissues. PvGF14b was highest expression in root and lowest in immature leaf. Whereas PvGF14c and PvGF14f showed tissue-specific expression. PvGF14c was mainly expressed in immature and mature leaves. PvGF14f was highest expression in mature leaves. These four genes were not significantly differentially expressed in mature leaf before bamboo flowering and during flower development. PvGF14b and PvGF14c were not induced by circadian rhythm. PvGF14c displayed subcellular localization in the cytoplasm and PvFT in nucleus and cytoplasm. Yeast two-hybrid screening and bimolecular fluorescence complementation confirmed the interaction between PvGF14c and PvFT. The overexpression of PvGF14b, PvGF14c, and PvGF14e significantly delayed flowering time in transgenic Arabidopsis under long-day condition. These findings suggested that at least three PvGF14 genes are involved in flowering and may act as a negative regulator of flowering by interacting with PvFT in bamboo.
In higher plants, flowering transition time is a major factor affecting the adaptability of plants to their environment. Flowering time in higher plants are controlled by complex signaling pathways, such as photoperiod, autonomous flowering, vernalization, and gibberellin pathways, which are independent yet interconnected with one another, eventually forming a complex network (Johanson et al., 2000; Kim et al., 2009). SUPPRESSOR OF OVER-EXPRESSION OF CONSTANS1 (SOC1), FLOWERING LOCUS T (FT), and LEAFY (LFY) are considered integrating factors in these pathways (Moon et al., 2005) and are located downstream of FLOWERING LOCUS C (FLC) and CONSTANS (CO) genes, which regulate plant flowering time by integrating different flowering signals. The FT protein, which is synthesized in leaf blade and migrated along phloem tissue, plays a critical role in the shoot apical meristem (SAM). FT binds to 14-3-3 protein, and then interacts with transcription factor, FLOWERING LOCUS D (FD), thereby promoting the flowering of plants (Taoka et al., 2011). Whereas, overexpression of OsGF14c, an isoform of the rice 14-3-3 gene family, delayed strongly flowering time (Purwestri et al., 2009).
In eukaryotes, the 14-3-3 protein family comprises highly conserved proteins that exist as multiple isoforms in many species. Seven 14-3-3 genes are present in mammals (Arner and Holmgren, 2006), eight in rice (Chen et al., 2006), and 13 in Arabidopsis (Yao et al., 2007). According to homologous sequence evolution analysis, 14-3-3 proteins in plants can be divided into 𝜀 and non-𝜀 groups: the latter is plant specific (Chung et al., 1999; DeLille et al., 2001; Ferl et al., 2002). The members of the 14-3-3 family exhibit different expression patterns and appear to have varied functions. For example, BdGF14d, a non-𝜀 14-3-3 gene in Brachypodium distachyon, can enhance salt stress tolerance (He et al., 2017). OsGF14b, a non-𝜀 14-3-3 protein in rice, positively regulates panicle blast resistance while negatively regulating leaf blast resistance (Liu Q. et al., 2016). Whereas, overexpression of OsGF14c negatively regulate flowering time in rice (Purwestri et al., 2009).
Previous studies showed that 14-3-3 proteins are involved in the regulation of flowering in many plants (Mayfield et al., 2007; Paul et al., 2008; Purwestri et al., 2009). They interact with FT in Arabidopsis and SP (SELF-PRUNING, ortholog of FT) in tomato (Pnueli et al., 2001; Zhang, 2012). In rice, 14-3-3 proteins act as intracellular receptors for florigen Hd3a (Heading date 3a), an ortholog of Arabidopsis FT (Taoka et al., 2011). Hd3a is localized in the cytoplasm and nucleus, whereas OsGF14c is mainly found in the cytoplasm. Hd3a–GF14c interaction is mainly detected in the cytoplasm, indicating that Hd3a cytoplasmic retention is increased by interacting with GF14c (Taoka et al., 2011). Thus, overexpression of OsGF14c plants display a delayed flowering phenotype, suggesting that OsGF14c acts as a negative regulator of flowering by interacting with Hd3a (Purwestri et al., 2009).
Similar to rice, bamboo plants belong to Gramineae but exhibit different flowering characteristics. The flowering cycle of bamboo generally lasts 3–120, and most kinds of bamboo undergoing flowering die (de Carvalho et al., 2013). However, the cause of bamboo flowering remains unclear. Phyllostachys violascens sprouts in early stages, produces high yields, provides good taste, offers remarkable economic benefits. As such, this species is well known for shoot production in China. P. violascens belong to sporadically flowering bamboos, which exhibit either a flowering with dying pattern or a flowering without dying pattern. The flowering of bamboo leading to death reduces the production of bamboo shoots and causes economic loss. Thus, many research groups tried to uncover the flowering mechanism of bamboo plants by genomes and transcriptomes (Zhang et al., 2012; Peng et al., 2013; Gao et al., 2014; Shih et al., 2014; Zhao et al., 2014). In addition, numerous genes and miRNAs related to bamboo flowering were reported (Lin et al., 2010; Gao et al., 2015; Zhao et al., 2015; Guo et al., 2016a; Liu S. et al., 2016; Zheng et al., 2017). Our group reported previously that PvFT played an important role in the induction of bamboo flowering (Guo et al., 2016b). However, no proteins interacting with PvFT have been identified in bamboo. In the present study, four non-𝜀 14-3-3 genes and their expression profiles in P. violascens were identified. The interaction of PvGF14c and PvFT in P. violascens were determined. Meanwhile, overexpression of PvGF14b, PvGF14c, and PvGF14e was shown to delay the flowering time of transgenic Arabidopsis under long-day (LD) condition.
Materials and Methods
Plant Materials
Phyllostachys violascens samples were harvested from the Bamboo Garden of the Zhejiang A & F University. To detect the expression of Pv14-3-3 during the flowering period, we collected leaf samples every 10 days from April 2 to May 31. According to our observation, the plants maintained flowering from mid-March to mid-May. The leaves from the flowering P. violascens were collected every 3 h for nine times to analyze the diurnal expression of the target genes. To determine the tissue-specific expression, we sampled mature leaves, immature leaves, stems, roots, and spikelets. All samples were stored at -80°C until further experiment. Wild-type (ecotype Columbia) and transgenic plants of Arabidopsis were cultivated in a controlled temperature room under 22°C with 16-h light/8-h dark cycle.
Isolation of Four Pv14-3-3 Genes
Total RNA was extracted from the leaves by using the Trizol method. First-strand cDNA was synthesized using a Super ScriptTM kit (Invitrogen, United States) according to the manufacturer's instructions. Genomic DNA was isolated by the modified CTAB method from leaves (Guo et al., 2016b). According to 14-3-3 homologous genes of rice, BLAST was performed against Phyllostachys heterocycla cDNA database (Peng et al., 2013), and eight Ph14-3-3 genes were identified. Of these genes, four candidate genes that shared more than 85% amino acid sequence identity with OsGF14c. Four primers used to amplify Pv14-3-3s were designed on the basis of four candidate genes of P. heterocycla (Table 1). The full-length cDNA and DNA sequences of four non-𝜀 Pv14-3-3s of P. violascens were amplified through PCR with the designed primers (Table 1). The thermocycling conditions were as follows: initialization at 94°C for 3 min; 32 cycles of denaturation at 94°C for 30 s, annealing at 50–60°C, and extension at 72°C for 50 s; and final extension at 72°C for 10 min. The amplified fragments were separated through electrophoresis on 1.5% (m/v) agarose gel and stained with ethidium bromide.
Bioinformatics Analysis
The open reading frames of four Pv14-3-3s were confirmed using the ORF Finder1. The exon–intron organization of four Pv14-3-3s was revealed using a gene structure display server2. The protein domains of four Pv14-3-3s genes were predicted using InterPro3. The Molecular weights and isoelectric points of 14-3-3 proteins were predicted using ExPASy program4. The phylogenetic tree of 14-3-3 proteins from P. violascens, P. heterocycla, Arabidopsis, and Oryza sativa was constructed using the neighbor-joining method in MEGA 5.05 with bootstrap values obtained from 1000 replications.
Expression Analysis of Four Pv14-3-3s Genes
Total RNAs were extracted from the collected samples by using the Trizol method. First-strand cDNA was synthesized using a Super ScriptTM kit (Invitrogen, United States). The qRT-PCR primers were designed according to the non-conserved region of full-length cDNA sequences of four Pv14-3-3s. The NTB gene was used as the internal control gene (Table 1; Fan et al., 2013). The qRT-PCR analysis was performed using CFX96TM Real-Time PCR Detection System (Bio-Rad). Reactions were performed in 20-μl mixtures consisting of 10 μl 2 × SYBR Premix Ex Taq II Mix (Takara), 0.5 μl each of forward or reverse primer, 0.5 μl cDNA template (100 ng/μl), and 8 μl double distilled H2O. The program was 95°C for 30 s, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s. The data were analyzed by the 2-ΔΔCt method. Three independent replicates were performed for each experiment.
Subcellular Localization
The full-length sequence of PvGF14c or PvFT were inserted at the C- and N-terminus of CaMV35S-GFP vector, forming PvGF14c-GFP or PvFT-GFP fusion protein, transient expression. The transient expression of the PvGF14-GFP or PvFT-GFP fusions in onion epidermal cells was made by the particle bombardment method (Wang et al., 1988). A confocal laser scanning microscope was used to observe the onion epidermal cells.
Plasmid Construction and Transformation
The full-length ORF of Pv14-3-3s was attached to CaMV35S-GFP to form the plasmid 35S::Pv14-3-3s. Afterward, the floral dipping method mediated by Agrobacterium tumefaciens strain GV3101 was performed to convert the plasmid into wild-type Arabidopsis plants. The transgenic Arabidopsis seeds were chosen using 50 mg/ml hygromycin in 0.5× solid medium. Positive transgenic plants were further identified through PCR. Flowering time was measured using lines with typical phenotypes. The expression level of four Pv14-3-3s genes in transgenic and WT Arabidopsis were analyzed by qRT-PCR using gene-specific primers (Table 1).
Yeast Two-Hybrid (Y2H) Assay
The yeast two-hybrid (Y2H) assay was conducted following a previously reported method (Sun et al., 2015). The ORF of PvFT and PvGF14c were respectively cloned into the pGBKT7 BD vector and pGADT7 AD eukaryotic expression vectors for the swapping experiment. The Y2H was performed according to the BD Matchmaker Library Construction & Screening Kits instructions (Clontech, Palo Alto, CA, United States).
Bimolecular Fluorescence Complementation
The ORF sequence of PvGF14c was inserted into pSAT4-cEYFP-C1-B (nYFP) vector. The ORF sequence of PvFT or AtFT was inserted into pSAT1 nEYFP - C1 (cYFP) vector, respectively. Subsequently, these vectors were introduced into DH5α. And then, the purified plasmids were introduced into the onion epidermal cells following the protocol in Section "Subcellular Localization."
Statistical Analysis
Statistical analysis was performed using SPSS 19.0 (SPSS Inc., Chicago, IL, United States). Differences were analyzed with one-way ANOVA followed by Tukey's range test. Significance was accepted at the level of P < 0.01.
Cloning of Four Pv14-3-3 Genes in P. violascens
We performed a BLAST 14-3-3 homologous genes of rice against P. heterocycla cDNA database and identified eight Ph14-3-3 genes named PhGF14a–h according to the nomenclature of rice. PhGF14a–h showed 77, 88, 96, 83, 91, 86, 58, and 44% homolog to OsGF14c, respectively (Table 2). Phylogenetic analysis showed that P. heterocycla contained two 𝜀 and six non-𝜀 14-3-3 isoforms (Figure 1A). Four candidate genes shared more than 85% of amino acid sequence identity with OsGF14c. Four primers for cloning Pv14-3-3s were designed according to four candidate genes of P. heterocycla, and four Pv14-3-3s genes from P. violascens were cloned and named PvGF14b, PvGF14c, PvGF14e, and PvGF14f, respectively. All of information about four non-𝜀 Pv14-3-3 genes is shown in Table 3. The four Pv14-3-3 proteins contain 256–262 amino acid residues, and their molecular weight range was from 28.9 to 29.8 kDa. Their isoelectric points were from 4.70 to 4.86. The exon–intron structural analysis reveals that four non-𝜀 Pv14-3-3 genes contain five exons and four introns (Figure 1B). To understand the genetic relationships of Pv14-3-3s in plants, we performed a phylogenetic analysis of Arabidopsis, O. sativa, P. violascens, and P. heterocycla (Table 4). The results indicated that Pv14-3-3s were closely related to Ph14-3-3s, as revealed by their occurrence in the same branch (Figure 1A), and four Pv14-3-3s genes belonged to the non-𝜀 group. Furthermore, we performed a multiple sequence alignment of the amino acid sequence of four Pv14-3-3s genes and demonstrated that four 14-3-3 proteins of P. violascens were highly conserved. Some sequences were completely conserved among four proteins. Eight amino acid residues in the N-terminal and 18 amino acid residues in the C-terminal exhibited large differences, suggesting that the various termini could provide different functions in each protein by interacting with a series of target proteins. Four non-𝜀 Pv14-3-3 proteins in P. violascens consisted of the following nine antiparallel α-helices: αC and αD have the longest sequence of amino acids with 30–34 amino acid residues; αA–D form dimers at the interface; αC, αE, αG, and αL constitute conservative peptides with a structure similar to 14-3-3s in other plants (Figure 1C).
FIGURE 1. Phylogenetic and gene structure analyses of Pv14-3-3s. (A) Phylogenetic tree of 14-3-3s from P. violascens, P. heterocycla, O. sativa, and Arabidopsis plants constructed using MAGA5.0. The registration number 14-3-3 family genes were shown in Table 4. (B) Gene structures of Pv14-3-3s. [scale=0.30]img001 represent the exons; [scale=0.80]img002 represent the introns. (C) Amino acid sequence alignments of four Pv14-3-3s.
Analysis on the Expression Feature of Four non-𝜀 Pv14-3-3s of P. violascens
To examine the biological functions of four non-𝜀 Pv14-3-3s genes, qRT-PCR was performed to analyze the temporal expression and tissue-specific expression of 14-3-3 genes of P. violascens. The results showed that PvGF14b and PvGF14e were constitutive expression, whereas PvGF14c and PvGF14f exhibited tissue specificity (Figures 2A–D). The highest expression level of PvGF14f was observed in the stem. Its expression was undetectable in the immature leaves and in the roots (Figure 2D). The highest expression level of PvGF14c was found in the leaves. Conversely, a low expression level was recorded in the root, stem, and spikelet (Figure 2B). The samples of mature leaves collected were used to detect the expression levels of four Pv14-3-3s genes before bamboo flowering and during flowering development. The expression levels of each Pv14-3-3s genes did not significantly different between these two development stages (Figures 2E–H). Before bamboo flowering, the relative expression level of three Pv14-3-3s genes, PvGF14b, PvGF14c, and PvGF14f were similar and higher than it of PvGF14e. Furthermore, the data showed that PvGF14b and PvGF14c were expressed in 24 h with highest at 11 am, but without obvious difference between day and night, suggesting that PvGF14b and PvGF14c were not shown to have diurnal rhythm expression (Figures 2I,J).
FIGURE 2. Expression analysis of four Pv14-3-3 genes in P. violascens. (A–D) Relative expression of four Pv14-3-3 genes in different tissues. (E–H) Temporal expression of four Pv14-3-3 genes in leaves during different development stages. The period between black solid lines represents flowering time. (I,J) Daily expression of PvGF14b and PvGF14c. Dusk and dawn are at 17:00 and 5:30. Gray areas indicate darkness. Gray shading denotes the dark/night portion of 24 h cycle. Expression of the PvNTB gene was used as an internal control. Error bar indicates standard deviation. A, B indicate highly significant differences (P < 0.01). The image appeared below the part "J" is rends in daylength and temperature during the period of collecting samples.
The Influence of Ectopic Expression of Four Pv14-3-3 Genes on Flowering Time in Transgenic Arabidopsis
To demonstrate whether four 14-3-3 genes can influence flowering in plants, we constructed the overexpression vectors 35S::PvGF14b, 35S::PvGF14c, 35S::PvGF14e, and 35S::PvGF14f which were transformed to Arabidopsis, respectively, and got 31 T1 GF14b-ox plants, 42 T1 GF14c-ox plants, 17 T1 GF14e-ox plants, and 15 T1 GF14f-ox plants. At last, four independent lines of GF14b-ox, GF14e-ox, and GF14f-ox in the T2 generation, as well as four independent lines of GF14c-ox in the T3 generation were grown under greenhouse condition for further analysis. 35S::PvGF14b, 35S::PvGF14c and 35S::PvGF14e delayed flowering in transgenic Arabidopsis (Figures 3–5). Whereas, PvGF14f-overexpressing plants showed the similar flowering time as that of the wild-type (Figure 6). The number of rosette leaves of PvGF14b, PvGF14c, and PvGF14e transgenic plants was 2–5, 5–7, and 3–6 more than that of the wild-type, respectively (Figures 3–5). The bolting time in 35S::PvGF14b, 35S::PvGF14c and 35S::PvGF14e transgenic Arabidopsis plants was 3–4, 6–8, and 4–6 days later than that in the wild-type (Figures 3–5).
FIGURE 3. Phenotype analysis of 35S::PvGF14b Arabidopsis plants under LD conditions. (A) Phenotype differences in flowering between 35S::PvGF14b transgenic plants and wild-type plants. Photographs were taken at 26-day-old wild-type plants. (B) The number of rosette leaves in T2 35S::PvGF14b in transgenic plants (n = 20) and wild-type plants. (C) Bolting time in T2 35S::PvGF14b transgenic plants (n = 20) and wild-type plants. (D) qRT-PCR expression analysis of PvGF14b. Asterisks "∗∗" indicate significant differences in comparison with the WT at P < 0.01.
FIGURE 4. Phenotype analysis of 35S::PvGF14c Arabidopsis plants under LD conditions. (A) Phenotype differences in flowering between 35S::PvGF14c transgenic plants and wild-type plants. Photographs were taken at 28-day-old wild-type plants. (B) The number of rosette leaves in T3 35S::PvGF14c in transgenic plants (n = 24) and wild-type plants. (C) Bolting time in T3 35S::PvGF14c transgenic plants (n = 24) and wild-type plants. (D) qRT-PCR expression analysis of PvGF14c. Asterisks "∗∗" indicate significant differences in comparison with the WT at P < 0.01.
FIGURE 5. Phenotype analysis of 35S::PvGF14e Arabidopsis plants under LD conditions. (A) Phenotype differences in flowering between 35S::PvGF14e transgenic plants and wild-type plants. Photographs were taken at 25-day-old wild-type plants. (B) The number of rosette leaves in T2 35S::PvGF14e in transgenic plants (n = 20) and wild-type plants. (C) Bolting time in T2 35S::PvGF14e transgenic plants (n = 20) and wild-type plants. (D) qRT-PCR expression analysis of PvGF14e. Asterisks "∗∗" indicate significant differences in comparison with the WT at P < 0.01.
FIGURE 6. Phenotype analysis of 35S::PvGF14f Arabidopsis plants under LD conditions. (A) Phenotype differences in flowering between 35S::PvGF14f transgenic plants and wild-type plants. Photographs were taken at 27-day-old wild-type plants. (B) The number of rosette leaves in T2 35S::PvGF14f in transgenic plants (n = 20) and wild-type plants. (C) Bolting time in T2 35S::PvGF14f transgenic plants (n = 20) and wild-type plants. (D) qRT-PCR expression analysis of PvGF14f.
To determine whether the flowering time in transgenic Arabidopsis had relation with the expression of examined genes, we detected the transcript levels of PvGF14b, PvGF14e, and PvGF14f in T2 lines of 35S::PvGF14b, 35S::PvGF14e, and 35S::PvGF14f Arabidopsis, as well as PvGF14c expression level in T3 lines of 35S::PvGF14c Arabidopsis, respectively. The data revealed that expression levels of examined genes in different transgenic lines were significantly increased (Figures 3–6). The flowering time was positively associated with the expression of PvGF14c in transgenic Arabidopsis plants (Figure 3D). However, there was no correlation between other three PvGF14s mRNA abundance and the flowering time (Figures 4D, 5D, 6D).
Subcellular Localization of PvGF14c
Previous studies showed that OsGF14c predominantly localized in the cytoplasm and OsGF14c-ox plants were delayed by 5–20 days in flowering relative to wild-type plants (Purwestri et al., 2009). In addition, based on the data above, we found that the effect of PvGF14c on flowering time of transgenic plants was stronger than other three PvGF14s genes (Figures 3–6) and phylogenetic tree showed that PvGF14c was closest related to OsGF14c (Figure 1). Therefore, we built PvGF14c-GFP fusion constructs driven by CaMV35S promoter to analyze the intracellular localization of PvGF14c and to investigate the molecular mechanism of PvGF14c. These constructs were introduced into onion epidermal cells for transient expression. As expected, the empty GFP signals were ubiquitously distributed throughout the cells (Figure 7A). PvGF14c-GFP fusion proteins were localized in the cytoplasm (Figure 7B).
FIGURE 7. Subcellular localization of PvGF14c and PvFT protein. (A) Cells bombarded with 35S::GFP. (B) Cells bombarded with PvGF14c-GFP. (C) Cells bombarded with PvFT-GFP.
Interaction between PvGF14c and PvFT Proteins
Previous presentation showed that the binding of 14-3-3 to Hd3a is important for flowering and OsGF14c interacted with Hd3a by Y2H screen and bimolecular fluorescence complementation (BiFC; Purwestri et al., 2009; Taoka et al., 2011). Thus, Y2H screening was performed to identify the relationship between PvGF14c and PvFT. The results demonstrated that PvGF14c could interact with PvFT protein as either a bait or a prey in P. violascens (Figure 8). Meanwhile, we used BiFC to determine the interaction of PvFT and PvGF14c in onion cell. As shown in Figure 9, the PvFT and PvGF14c interaction was detected in the cytoplasm. However, PvFT alone was detected in cytoplasm and nucleus (Figure 7C).
FIGURE 8. Identification of the interaction between PvGF14c protein and PvFT protein in yeast. The interaction between pGBKT7-53 and pGADT7-T was the positive protein–protein control, whereas the interaction between pGBKT7-lam and pGADT7-T was the negative protein–protein control.
The PvGF14c and AtFT Complex Is Localized in the Cytoplasm
Purwestri et al. (2009) presented that the interaction of Hd3a and OsGF14c resulted in Hd3a retaining in cytoplasmic and delayed flowering time in OsGF14c-ox. Our data showed that PvGF14c-ox plants also displayed the delayed flowering phenotype. Therefore, we determined the distribution of PvGF14c and AtFT in vivo by using BiFC. As shown in Figure 9, the PvGF14c and AtFT complex was located in cytoplasm.
Plant 14-3-3 proteins act in a phosphorylation network and play an important role in plant growth and development. 14-3-3 Proteins interact with FT in Arabidopsis, with SP in tomato, and Hd3a in rice (Pnueli et al., 2001; Purwestri et al., 2009; Zhang, 2012). Studies on overexpression transgenic plants and mutant plants have revealed that OsGF14c functions as a negative regulator of flowering (Purwestri et al., 2009). However, the exact role of 14-3-3 proteins in the regulation of flowering in P. violascens has not yet to be reported.
In this study, four 14-3-3 genes, which share more than 85% amino acid sequence identity with OsGF14c, were identified in P. heterocycla and synthesized from P. violascens. The molecular masses and isoelectric points of four Pv14-3-3 proteins were consistent with the values of 14-3-3 families in reported plants (Xu and Shi, 2006; Li and Dhaubhadel, 2011; Qin et al., 2016). In Arabidopsis, the non-𝜀 group comprises four exons and three introns, introns insert position quite conservative (Wu et al., 1997). By comparison, the 𝜀 group generally includes six to seven exons, and the genetic structures of the 𝜀 group are different from those of the non-𝜀 group (DeLille et al., 2001). But the exon–intron structure analysis revealed that the four Pv14-3-3s consisted of five exons and four introns (Figure 1B), which are similar to non-𝜀 14-3-3s in rice (Chen et al., 2006).
Phylogenetic analysis indicated that four Pv14-3-3s proteins all belonged to the non-𝜀 group. Pv14-3-3s were closely related to Ph14-3-3s, and they gathered in the same branch. Meanwhile, Pv14-3-3s were closely related to non-𝜀 14-3-3s of O. sativa. The conservation of non-𝜀 14-3-3s between P. violascens and O. sativa indicated that the functions of four non-𝜀 Pv14-3-3s may be similar to those of non-𝜀 14-3-3s in O. sativa. Especially, PvGF14c is the highest identity with the OsGF14c which act as intercellular receptor for florigen, and OsGF14c-overexpressing plants display a delay flowering phenotype (Purwestri et al., 2009; Taoka et al., 2011). Thus, the regulation of flowering by Pv14-3-3s in P. violascens should be further investigated.
To examine the biological functions of four Pv14-3-3s, we conducted temporal-spatial expression analysis and observed that the expression pattern of these four genes were diverse, and there were various degree of expression in leaf. The mRNA of PvGF14c accumulated strongest in leaf relative to other tissues (Figure 2B). In addition, their expression levels did not differ before bamboo flowering and during flowering development (Figures 2E–H). The expression of PvGF14c and PvGF14b showed no diurnal changes, suggesting that the expression of these two genes were independent of photoperiod during plant development (Figures 2I,J).
In rice, GF14c-overexpressing plants displayed late flowering time and the knockout mutants exhibited early flowering under short-day conditions (Purwestri et al., 2009). In our study, PvGF14c-overexpressing Arabidopsis plants underwent late flowering under LD conditions, which is similar to OsGF14c delaying strongly flowering time (Purwestri et al., 2009). Considering that the 14-3-3 protein family were highly conserved, we also determine whether three other 14-3-3 proteins could regulate flowering. The results showed that PvGF14b and PvGF14e overexpression delayed slightly flowering time in transgenic Arabidopsis. Whereas, PvGF14f-ox plants showed the similar flowering time as that of the wild-type (Figures 3–6).
14-3-3 Isoforms bind to many kinds of proteins including regulators of flowering time, such as CO (Mayfield et al., 2007), and florigen FT (Pnueli et al., 2001). In rice, 14-3-3 proteins act as intercellular receptors for Hd3a which is localized in the cytoplasm and nucleus, whereas GF14c are mainly found in the cytoplasm. Hd3a–GF14b and Hd3a–GF14c interactions were detected mainly in the cytoplasm (Taoka et al., 2011). In wild-type of rice, Hd3a protein transported from leaves to the SAM is first received by OsGF14c protein in the cytoplasm, and then Hd3a–14-3-3 complex is translocated to the nucleus, where it forms a larger protein complex, named florigen activation complex, with OsFD1, then induces transcription of OsMADS15, leading to flowering (Taoka et al., 2011, 2013). 14-3-3 Proteins regulate their interaction proteins through a variety of mechanisms such as altering their subcellular localization (Roberts, 2003). The delayed flowering phenotype in GF14c-ox plants can be explained by the increased cytoplasmic retention of Hd3a by OsGF14c which are highly expressed in plants (Purwestri et al., 2009; Taoka et al., 2011). In our study, PvGF14s-ox plants exhibit delayed flowering. The interaction of PvGF14c and PvFT in P. violascens as well as PvGF14c and AtFT complex locating in cytoplasm were determined. These experiments hinted that high expression of PvGF14c gene regulate the import of FT into the nucleus in transgenic Arabidopsis. The floral induction could be attenuated by the cytoplasmic retention of FT by PvGF14s.
In flowering regulation, whether Pv14-3-3 proteins bind to PvFT and PvFD, forming a complex, and participate in the molecular machinery of flowering regulation is unknown, and the molecular mechanism of PvGF14-3-3s in regulating flowering should be further investigated.
Author Contributions
XG, BL, and GX conceived and designed the experiments. BL, GX, ZW, BM, and XL conducted and analyzed the experiments. KL helped to analyze the data. BL and XG wrote the paper. All authors read and approved the manuscript.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was supported by the following grants: National Natural Science Foundation of China (grant no. 30901155), Natural Science Foundation of Zhejiang Province (grant no. Y307499).
- ^ http://www.ncbi.nlm.nih.gov/gorf
- ^ http://gsds.cbi.pku.edu.cn
- ^ http://www.ebi.ac.uk/
- ^ http://web.expasy.org/compute_pi
- ^ www.megasoftware.net
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Keywords: Phyllostachys violascens, Pv14-3-3s, PvGF14c, flowering, ectopic expression
Citation: Li B, Xiao G, Luo K, Wang Z, Mao B, Lin X and Guo X (2018) Overexpression of PvGF14c from Phyllostachys violascens Delays Flowering Time in Transgenic Arabidopsis. Front. Plant Sci. 9:105. doi: 10.3389/fpls.2018.00105
Received: 18 October 2017; Accepted: 19 January 2018;
Published: 14 February 2018.
Edited by:Sagadevan G. Mundree, Queensland University of Technology, Australia
Reviewed by:Hak Soo Seo, Seoul National University, South Korea
Keqiang Wu, National Taiwan University, Taiwan
Copyright © 2018 Li, Xiao, Luo, Wang, Mao, Lin and Guo. 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) and the copyright owner 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: Xiaoqin Guo, email@example.com
† These authors have contributed equally to this work.

subset: commoncrawl


text:
The South Street Arts Centre in Katesgrove, Reading throw open their doors from 3pm on Saturday 23 November for their Beat Connection 'all-day party'.
The bands playing at the event include JOHN, TVAM, Talk Show and YOWL.
Promoters Heavy Pop add:
Heading up the cream of local talent are Apple Music approved alt-rockers Mellor; skuzzy punk 'n' rollers Workin' Man Noise Unit; lo-fi drums and synth soloist Despicable Zee; and alternative pop trio Chalk City. Also playing the on the day are psychedelic shoegazers Doops; noise rock three-piece Mythical Ash Cloud; drone-doom duo Cody Noon and psych-rock newcomers Meanwhile In Bat Country.
Food will be available from Tutu's Ethiopian Table and vegetarian food from We Are Friends. Beat Connection is open to people 14 years or older and tickets can be purchased online. If you would like to cover the event or interview some of the artists for the Whitley Pump, please get in touch with us at [email protected].
There are music videos from many of the bands on the Heavy Pop website.
Video (c) 'Meanwhile in Bat Country' via Youtube.
- South Street Arts Centre
- Beat Connection on Facebook and tickets
- Heavy Pop


text:
report contains information on automotive vehicle production in Russia, particularly passenger cars and commercial vehicles
Ford Mondeo sedan has received a technological upgrade aimed at the safety improving of the car and improving of the intelligent driver assistance systems. People can order updated Ford Mondeo at all official Ford dealers in Russia.05 julymore info
According to the assessment of the analytical agency AUTOSTAT, KIA Rio remains the most popular new passenger car with automatic transmission in Russia. For 6 months of 2019, 34.6 thousand units with automatic transmission were sold - 5.7% higher than in January – June'18.
The company "Foton Motor" announced the sale of Foton Sauvana cars of 2016 with the benefit of up to 390 thousand rubles. According to the press service of Foton Motor, the maximum benefit is achieved through discounts on the trade-in system (from 100 to 150 thousand rubles, depending on the equipment), as well as discount when buying a car for leasing (at a rate of 3%). As a result, the Sauvana SUV of 2016 can be purchased at a price of 1 million 311 thousand 340 rubles.
In addition, until June 30th, 2018, the action to provide an extended 7-year warranty for Foton Sauvana continues. Thus, for all Foton Sauvana cars purchased in the period from March 10th, 2017 to June 30th, 2018, an extended warranty for the engine and transmission parts are provided - 7 years or 120 thousand km of mileage, whichever comes first.
Foton Sauvana is a large frame SUV with dimensions of 4830 × 1910 × 1885 mm and a ground clearance of 220 mm. The car is offered with a petrol turbocharged 4G20TI engine with a displacement of 2.0 liters in two power versions - 201 or 217 hp. The design of Sauvana uses the components of the world's leading manufacturers - automatic transmission ZF and mechanical Aisin, transfer case Borg Warner with three modes of operation, rear axle Dana. All Foton Sauvana vehicles are equipped with an emergency response system for ERA-GLONASS in case of accidents.
|Source: www.autostat.ru


text:
1. Why should the LED be protected?
White LED because there are many advantages, are more and more into people's daily life, its use is now becoming very great. It is a new device that has its own use on the features. White LED is a voltage-sensitive device. Each LED work when the current should not exceed 20mA, more than too much LED will be easily burned. If the LED is normal use, its life is very long. But people in the actual use of LED is often easy to bad, what reason? In fact, is not taking into account the use of LED features and add protection circuit.
LED is a photoelectric semiconductor device, in the assembly process is easy to be static injury. This requires electrostatic protection during assembly. We found that many manufacturers do not have this concept or do not understand, this is not enough.
LED in the actual work is 20mA current for the upper limit, but often due to various reasons in the use of the current increase, if not take protective measures, this increase in current over a certain time and amplitude after the LED It will be damaged.
2. Cause of LED damage
(1). Sudden increase in supply voltage. So the power supply voltage suddenly rose a lot of reasons, such as the quality of power problems, or improper use of the user, etc. may cause the power supply voltage suddenly increased.
(2). A short circuit of a component or printed line or other wire in the line forms a local short circuit of the LED power supply path, which increases the voltage in this area.
(3). An LED because of its own quality damage and thus short-circuit, its original voltage drop on the other LED passed on.
(4). The temperature inside the lamp is too high, so that the characteristics of LED deterioration.
(5). The interior of the lamp enters the water and the water is conductive.
(6). In the assembly time did not do anti-static work, so that the internal LED has been static damage. Although the application of the normal voltage and current values, but also easily lead to damage to the LED.
These reasons will cause a significant increase in LED current significantly, and soon the LED chip will be burned because of overheating. According to our experience, most of the LED burned after the two-pole short circuit, a small part of the open circuit. Each LED about 3.2V or so the pressure drop, it burned after the explosion if this string of LED will not light up. If the short circuit of this voltage to the other LED, resulting in other LED current, the other LED will be burned faster, and even critical power. Originally small damage is very easy to cause a major accident. LED is generally installed in the high, the installation is not easy, it is more difficult to repair. So the protection of LED is the actual demand, but there is no attention, but also a lot of people no way to deal with the dilemma.
How to protect the LED
The protection of the LED we first thought is to use the fuse, but the fuse is a one-time, and the reaction speed is too slow, both the actual effect is also very troublesome, so the fuse is not suitable for the current LED lights finished, Because the LED lights are now mainly in the city's glorious works and lighting works. In response to this practical needs, we have done a lot of experiments, and according to the requirements of the project summed up the LED protection circuit to have the characteristics of it is very harsh: beyond the normal use of current can immediately start the protection, so that LED power supply path It is broken, so that LED and power can be protected in the normal light after the normal power supply can be restored, does not affect the LED work, the key is because it is a civilian product, the added circuit can not be too complex volume can not be too much, The cost is low. These requirements are contradictory, mutual constraints, it is difficult to achieve.
First, you should determine which protection circuit and protection device is selected.
1. We can choose to use the transient voltage suppression diode (referred to as TVS). Transient voltage suppression diodes are a high-performance protection device in the form of diodes. When its poles are subjected to a reverse transient high-energy impact, the high resistance between the two poles can be reduced to a low resistance at a rate of 10 times the negative time of 10 times the square, absorbing up to several kilowatts of surge power , The voltage between the two poles in a predetermined voltage value, the effective protection of the electronic circuit in the precision components. Transient voltage suppression diode has the advantages of fast response time, high transient power, low leakage current, good consistency of breakdown voltage, easy control of clamping voltage, no damage limit, small size and so on.
But found in actual use is not very satisfactory. It is not easy to find a TVS device that meets the required voltage. TVS devices are mainly used in lightning protection and lightning protection, and more than 220V over-voltage absorption, etc., and LED lamp power supply voltage is generally 24V or 12V, this voltage value of TVS finished very little, the test is not good. At the same time we know: LED light bead damage is mainly because the current is too large to make the chip caused by overheating. TVS can only detect overvoltages can not detect overcurrent. Overvoltage is certainly the cause of overcurrent, but it is difficult to choose the appropriate voltage protection point, this device can not be produced is difficult to use in practice.
Hot products:IP65 panel light,120cm linear lamp,150W power high bay,Ceiling mounted light,LED linear module lighting system,Linear pendant high bay,motion sensor linear lamp


text:
Meet the incredibly talented team behind Yellow Bliss Road! They are working either behind the scenes or front and center sharing new recipes with you each month. Be sure to check them out!
Julie | Bread Booze Bacon
Julie blogs at Bread Booze Bacon and is a self-proclaimed food nerd & cocktail enthusiast. She's totally obsessed with carbs, love Guinness, whiskey, and cheese. She could put bacon in just about everything, is a music nerd, loves "that's what she said" jokes, and playing superheroes with her boys.
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Fiona blogs over at Just So Tasty where she shares easy, classic recipes for desserts & sweet treats. A peanut butter lover, chocolate connoisseur, and sprinkle addict – she hopes to make your life sweeter, one cookie at a time. She also teaches yoga, because life is all about balance.
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Erica is a 30-something, Florida-bred foodie with an eye for pretty photos and incurable wanderlust. She discovered her love of food at a young age, and learned everything she knows about life and the kitchen from her Nana. The Crumby Kitchen was launched in 2014, and with the culinary expertise of her husband, Abe, she makes it a place where nothing is off-limits: from easy savory meals to over-the-top decadent desserts for two, four, or a crowd!
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James's love of cooking took flight at an early age when he became his grandmother's culinary right hand, and was further fueled by jobs at various food establishments and eateries. After college, James worked as an equities and futures trader on Wall Street for 15 years before retiring, and realizing his higher purpose: creating beautiful recipes his grandma would be proud of, and sharing them with others via his blog, Sip and Feast.
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Ashley is the creator of Spoonful of Flavor. She loves creating easy, wholesome meals and believes in balancing clean eats with sweet treats. When she is not in the kitchen, you will find her traveling to foodie spots near and far.
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Kylee is the creator of Kylee Cooks. She focuses on simple, no-fuss, homemade recipes that help solve the question of what to make for dinner. Kylee loves creating recipes that use readily available ingredients and presents them in a friendly, easy-to-follow way! She lives in Arizona with her hot husband, 2 young sons and loves camping, quad riding and road trips. And bacon. She LOVES bacon.
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Garlic Parmesan Risotto
Best Homemade Beef Burgers
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Melissa Williams is the creator of Persnickety Plates where she shares simple, family friendly recipes with everyday ingredients. She especially loves to bake and rarely meets a cookie she doesn't like. This wife and mom of two girls aims to share recipes that are quick and easy because even though life is busy, there's always time for good food.
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Crockpot Honey Bourbon Chicken
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Karen | Fresh Coast Eats
Karen is the creator of Fresh Coast Eats where she shares delicious and easy recipes inspired by the four seasons and our beautiful Great Lakes. She is passionate about teaching others how to select quality seasonal produce and preparing it simply, so it's natural flavors shine. Karen is also a certified sommelier and loves to incorporate wine into her recipes as well as suggest perfect food and wine pairings.
Three of Karen's most popular recipes are:
Vegan Cabbage Soup
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Adrienne | Creative Team Manager
Adrienne is a technical Virtual Assistant and Project Manager who shares tips and tools for dealing with stress and exhaustion over at Square Peg Wellness. She also likes to read, cook new recipes, and watch movies (as long as they are not scary!) You can most often find her exploring the local bookstore or walking her 14-year-old black lab.
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text:
Yes that would work. You'd need additional electronics to drive the motor and heating element since the pins on an arduino can provide only a limited amount of current.
Which microcontroller to choose? Answered
Hi all,
I need to use a microcontroller for my project and am thinking of getting the arduino UNO R3 development board which composes of the ATmega328 microcontroller.
I have to connect a small motor, a 16x2 LCD display and a miniature heating element to the arduino. I would like to know if the UNO R3 is suitable for my project.
PS: I am very new and noob regarding microcontrollers
The forums are retiring in 2021 and are now closed for new topics and comments.
Hmm ok
How do I go about doing that?
Put some numbers on "heating element" and "motor" for current, voltage and power.
To add on:
3 push buttons will also be connected


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The changes to the policies on housing loans and Central Provident Fund (CPF) usage are helpful towards reducing the liabilities of home owners who take a housing loan, and increasing their retirement income in the long term (Rules on HDB housing loans, CPF usage updated, May 10).
However, the changes also add to home owners' stress in the short term. Furthermore, some home owners seeking to increase their retirement income by selling their flats may face challenges.
Under the updated policies, some flat applicants may now qualify for a lower housing loan compared with what they would have received under the former policies.
While young flat applicants with little CPF savings enjoy the maximum Housing Board loans when they purchase a Build-to-Order flat or a resale flat which they will not outlive, their peers who are in need of a flat urgently and have to settle for a resale flat which they would outlive would be eligible for only a smaller HDB loan.
Similarly, the latter would also face restrictions with bank loans.
As a result, they would have to shell out more cash up front to make up the difference between the housing loan and the flat's price if they have insufficient CPF savings.
This would add to their burden in the short term, as some applicants may struggle to raise funds even though they would take on a smaller liability in the long run.
On the other hand, sellers of old flats may face difficulties when they try to monetise their homes to increase their retirement income.
With a shorter lease remaining on these flats, only home buyers who wish to utilise their CPF savings and apply for an HDB loan can be expected to be interested in them.
Young buyers with little CPF savings who wish to utilise their maximum HDB loan would be deterred from purchasing such flats.
Home owners of small older flats may also face greater challenges, as middle-income home owners with more savings seeking to upgrade their homes tend to prefer larger flats.
Timothy Toh


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Holiday order deadline is 12/10 — Free domestic shipping on stickers, cards, and orders of $40+*
This beautiful watercolor art print features vibrant pink and yellow flowers in the shape of Arizona. This print was created from an original watercolor painting by Melissa Balkon.
Usually ships in 2-5 days. Frame and mat not included.


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Hu Li Za Zhi. 2020 Feb ;67(1):44-54. PMID: 31960396
Abstract Title:
[Effect of Aromatherapy on Menopausal Symptoms, Heart Rate Variability, and Sleep Quality in Women].
BACKGROUND: Studies have shown that aromatherapy improve health problems related to anxiety, depression, heart rate variability (HRV), and sleep. However, the effect of aromatherapy in women who suffer from menopausal syndrome and its specific effects on HRV and sleep quality are unknown.PURPOSE: This study designed an aromatherapy intervention and evaluated its effect on menopausal syndrome, HRV, and sleep quality in women.METHODS: This double-blinded randomized controlled trial was conducted at a medical center hospital. A total of 84 participants who met the study criteria were randomly assigned using permuted block randomization. The experimental group received a 20-min inhalation of essential oil and the control group received a 20-min inhalation of sweet almond oil every night for 4 weeks. Posttest data was collected at 2 weeks after completion of the intervention. Data were collected using the Green Menopausal Symptom Scale, HRV device, and Pittsburgh Sleep Quality Index.RESULTS: After adjusting for age, the results of the generalized estimation equations (GEE) showed that all outcomes were significantly different in both the experimental group and the control group (p


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I like Linux, anime and video games like most people here. Please play vidya with me.
Looking at fake GBA carts' PCB and going feral over these misspelled Nintendo logos
I really wish Dynamax adventures showed you the last pokemon's type constantly or made it so you can see the entire map again.
Nobody seems to remember what type the legendary is so whenever I try to catch Kyogre it OHKOs everyone with surf because they didn't swap with water resists.
I don't want to make an introduction post so I'm just going to post pallas's cats again.
A cool community, I guess.


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- Slows Rapid Flashing Indicators
- Pre Wired
- Aluminium Heat Sink
- Single Pack
Narva LED Load Resistors prevent warning messages and rapid flashing indicators when used inline with LED tail lights. Supplied with 2 300mm wire tap connectors for easy installation and aluminium heat sink housing,
Technical Specifications
|Part Number
|24 Volt
We offer FREE tracked shipping Australia wide on all orders.Unless your purchase is a big bulky item you can expect it to be sent via Australia Post. A small number of our larger items are sent via courier.
All orders are dispatched from our centrally located warehouse in Adelaide, South Australia. Where possible your item will be picked, packed and shipped the same or next business day.
We offer a 30-day hassle free returns policy. If you choose to return an item simply contact our customer service team and we will supply you with a return postage label. See our full returns policy here.
All products sold by Tinker Wholesale Group come complete with a manufacturer warranty. Should you need to make a warranty claim please contact our customer service team and we will support you through the replacement / repair process.
Payment & Security
Your payment information is processed securely. We do not store credit card details nor have access to your credit card information.


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Very pieceful and quiet area with lots of trees, flowers and wildlife to enjoy. the apartment complex is very clean and well maimtaimed. units have personal balconies but in addition residents csn enjoy the ample open spaces with table chairs and a bbq to share.
"Find your new home at Paulson Park in Mountain View, CA. The highly-rated apartments are located in a quiet neighborhood. Be mindful of your security. Features like security cameras help to keep you safe. Apartments have newly renovated interiors for you to enjoy. Your vehicle is safe when you use the covered parking that is available to residents. Information on vacancies and lease options is available through property management. Call, or visit 111 Montebello Ave in Mountain View, CA."
Let me know when this property is back on the market
© 2020 RentPath, LLC. All rights reserved. All photos, videos, text and other content are the property of RentPath, LLC. APARTMENT GUIDE and the APARTMENT GUIDE Trade Dress are registered trademarks of RentPath, LLC or its affiliates.
Apple, the Apple logo, iPad, and iPhone are trademarks of Apple Inc., registered in the U.S. and other countries. App Store is a service mark of Apple Inc.
Information is copyrighted and school data provided by GreatSchools.org. Information is deemed reliable but not guaranteed. Schools listed above are based on distance from the selected location and are just a sampling of the schools in the area. To find a particular school district, and how it relates to a specific apartment, please contact the apartment manager.


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1. Grill El Asador
2. Everyman Cinema
3. London Waterloo Railway Station (WAT)
HISTORY UK: The main entrance to Waterloo Station is the Victory Arch, a memorial to employees of the London and South Western Railway staff killed in the First World War.
4. Green Park London Underground Station
Londonist: Next time you're walking the half-marathon between the Piccadilly and Jubilee lines at this station, keep an eye on the tiny wall tiles. Can you see what they've done? Clever.
5. Thames Ditton
HISTORY UK: There was a much-used river ford here, especially after Henry VIII took over Hampton Court Palace in 1529. The island later became popular with Victorian boating day-trippers.
6. The Establishment (Fulham)
Conor Ogle: Wine flights nice idea. Great burgers. Nice welcome.
7. Clapham South London Underground Station
HISTORY UK: The term 'man on the Clapham omnibus' to denote an average Londoner was coined by a journalist in the 19th century because Clapham was regarded as such an ordinary and unexceptional place.
8. Cavendish Road
HISTORY UK: Cavendish House once stood at the Clapham Common end of Cavendish Road. Here the scientist Henry Cavendish (d.1810) calculated the mass of the earth (about 6 trillion tons).
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- Join over 1.2 million students every month
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- Level: University Degree
- Subject: Historical and Philosophical studies
- Word count: 805
Plato and Descartes. In the works we studied by Plato (Apology, The Idea of the Good, and On the Shadows and Realities in Education) and Descartes (Meditations on the First Philosophy), both philosophers argue that we consist of a mind, or soul, which is
Extracts from this document...
Rick Serra March 6, 2012 Professor Bush CAL 105 Plato and Descartes Plato and Descartes, perhaps two of the greatest philosophers ever, have many similarities in their ways of thinking and reason but also have their differences. In the works we studied by Plato (Apology, The Idea of the Good, and On the Shadows and Realities in Education) and Descartes (Meditations on the First Philosophy), both philosophers argue that we consist of a mind, or soul, which is somehow connected with the body. Although both Descartes and Plato believe that the mind or soul can exist independently from the body, Descartes' mind-body argument, and Plato's soul-body argument, are quite different. In his third meditation Descartes comes up with the conclusion, "I am, I exist, is necessarily true each time it is expressed by me, or conceived in my mind." He comes to this conclusion from the two points within his argument that argue God exists. ...read more.
Consequently it will lose all its specific properties, yet it is still known as wax. Descartes argues that in order to comprehend what wax is, you must be able to know it in all its forms and predict its changes. Descartes claims that the shapes and forms that the wax could take are infinite. This means, one can only distinguish what an object is by understanding, rather than through sense perception. "I readily discover that there is nothing more easily or clearly apprehended than my own mind." Plato and Descartes both agree that there is a need reach the highest good or attain deeper spirituality by looking for reasons. Just like Descartes, Plato believes that in order to reach the right conclusion, people have to travel from perception to reason and understanding. The things that we perceive are actually mere shadows of the walls. ...read more.
Plato views knowledge as something that can be derived from distinct set of principles. "Reason answering to the highest, understanding to the second, faith (or conviction) to the third, and perception of shadows to the last." Unlike Descartes in which the groundwork was built on one's self, Plato believed knowledge rests upon Good as its foundation. The beliefs of Plato and Descartes are similar yet also differ in many ways. According to Plato, we understand and see shadows and not the real things. According to Descartes, one must also find what makes and object unique through reason, but he does this with a slightly different method. He disbeliefs what he feels is not undeniable and reconstructs his foundation of knowledge with himself as the base. Plato is not trying to doubt anyone's views but instead trying to expand one's knowledge of it. Both Descartes and Plato have the same ends, to try and reach the Good or God, the only thing that is different, is their method of achieving their goal. ?? ?? ?? ?? 1 Serra ...read more.
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Basic Training for Taxes for Military
Caroline Sposto
Basic Training for Tax Time for Military
Taxes are due on July 15, 2020, though if you're serving in a combat zone, you have additional time to file. Whether you have one month or several before your deadline, taking these steps can help you make sure you don't overpay.
Earned Income Tax Credits
See if you're eligible for any Earned Income Tax Credits (EITC).
Understand Your Combat Pay Exclusion
Money earned in combat zones recognized by the IRS, is usually tax exempt. You shouldn't have to do anything to receive the combat service exclusion, but if it isn't reflected on your W-2, contact your military pay office ASAP so they can make that correction and send you an amended W-2.
Know Which Income Is Taxable vs Non-taxable
In general, Taxable Income includes: non-combat pay wages, bonus, and incentive money, and student loan repayment from specific programs.
Non-taxable income includes:
- Allowances received for living and family expenses
- uniforms
- death expenditures
- moving and travel costs
- group-term life insurance
- professional education
Take Advantage of Travel & Lodging Deductions
If you travel more than 100 miles and stay overnight as a National Guard member or Armed Forces reservist, you can deduct those expenses – even if you don't itemize.
You can also deduct 50% of the cost of your meals while traveling, but be sure to not deduct more for food and lodging expenses than your per diem rate allows. Those rates are updated annually, and vary by state.
Deduct Any and All Non-reimbursed Moving Expenses
Even if you don't itemize. (Military moves don't have to meet the distance or time requirements of civilian moves before they're deductible.)
Decide to Take the Standard Deduction or Itemize
Determine your Standard Deduction and then figure out whether or not your legal itemized deductions add up to more. Choose the option that works in your favor.
Typical itemized deductions include:
- Home mortgage interest
- Property, state, and local income taxes, (and in some cases, sales tax)
- Medical and Dental expenses
- Charitable contributions
- Miscellaneous itemized deductions including the cost of purchase, cleaning, and maintenance of your military uniforms.
Decide Whether Keep Your Home Residency State
If you move for military reasons, you can keep your permanent legal residence in your home state, or choose the new state. Generally when you live in a state that collects income tax, but keep residency in a state that doesn't, you don't have to pay income tax in the new state. If income tax is withheld from your paycheck, you can file a nonresident return with that state to receive a refund.
Your Spouse Needs to Decide State Residency
Military spouses can either claim same state of legal residence as their partner, or claim the state they're living in. In certain cases they're allowed to claim the state of legal residence they had before marriage. Under the Servicemembers Civil Relief Act (SCRA) you won't be double taxed if your spouse works and is taxed in one state and has a permanent legal residence in another.
Important: SCRA doesn't apply to military children. If they're filing state income taxes, they need to file in the state where they live.
To decide which strategy works to your advantage, you'll need to compare state income tax rates.
The states with no personal income tax are: Alaska, Florida, Nevada, South Dakota, Texas, Washington, and Wyoming.
Tennessee and New Hampshire have no personal income tax on wages – though there is tax on interest and dividend income.
For more information on taxes, you can review this newly-revised IRS tax guide for members of the armed forces. Whether you're planning to file your taxes on your own, or with the help of a professional tax preparer, here are some additional tax information and tax preparation resources you'll find worthwhile.
- Resources for Military to Thrive this Tax Season
- Tip to Help Military Families Save on Taxes
- Free Tax Filing for Military
- State Benefits for Veterans (Including State Taxes)
- Top Personal Budget Plans
- 3 Basics of Budgeting for Military Families
About the author
Caroline Sposto is a writer, actor, and the founder of Savvy Civility, an educational company that specializes in civilian role play training. She has a passion for the arts, education, and small business.


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Jackson County is a
is inaccessible to the public.
The median income for a household in the county was $43,171, and the median income for a family was $50,970. Males had a median income of $38,919 versus $26,448 for females. The per capita income for the county was $20,171. About 6.50% of families and 9.00% of the population were below the poverty line, including 12.40% of those under age 18 and 6.10% of those age 65 or over.
In the county the population was spread out with 25.60% under the age of 18, 8.10% from 18 to 24, 30.40% from 25 to 44, 23.00% from 45 to 64, and 12.90% who were 65 years of age or older. The median age was 37 years. For every 100 females there were 104.20 males. For every 100 females age 18 and over, there were 103.70 males.
There were 58,168 households out of which 33.50% had children under the age of 18 living with them, 53.80% were married couples living together, 12.00% had a female householder with no husband present, and 29.80% were non-families. 24.60% of all households were made up of individuals and 9.60% had someone living alone who was 65 years of age or older. The average household size was 2.55 and the average family size was 3.03.
As of the census[10] of 2000, there were 158,422 people, 58,168 households, and 40,833 families residing in the county. The population density was 224 people per square mile (87/km²). There were 62,906 housing units at an average density of 89/sq mi (34/km²). The racial makeup of the county was 88.54% White, 7.92% Black or African American, 0.40% Native American, 0.53% Asian, 0.04% Pacific Islander, 0.83% from other races, and 1.74% from two or more races. 2.20% of the population were Hispanic or Latino of any race. 24.3% were of English, 21.7% of German,, 11.5% American, 9.9% Irish and 8.1% Polish ancestry according to the 2012 American Community Survey. 95.9% spoke English and 2.1% Spanish as their first language.
(information as of February 21, 2013)
The county government operates the county jail, maintains township roads, operates the major local courts, keeps files of deeds and mortgages, maintains vital records, administers public health regulations, and participates with the state in the provision of welfare and other social services. The county board of commissioners controls the budget but has only limited authority to make laws or ordinances. In Michigan, most local government functions – police and fire, building and zoning, tax assessment, street maintenance, etc. – are the responsibility of individual cities and townships.
When the South Branch and North Branch connect to form just the Kalamazoo River, it then flows through the rest of Calhoun County. It then enters Kalamazoo County and then Allegan County. When it reaches the towns of Saugatuck and Douglas it stops and enters Lake Michigan. In total the Kalamazoo River is 166 miles (267 km) long, and its watershed drains a total of 2,020 miles (3,250 km) and drains into 8 counties.
The South Branch starts in the wetlands near the town North Adams in Hillsdale County and flows through the rest of Hillsdale County. It enters Jackson County and goes through a small part of Hanover Township before reentering Hillsdale County and then reentering Jackson County where it goes through Pulaski Township. It then enters Calhoun County and connects with the Northern Branch when it reaches the town of Albion.
The North Branch starts in Jackson County in Hanover Township. It starts in Pine Hills Lake and Farewell Lake and flows through a small part of Liberty Township and then back into Hanover Township. It then flows into Spring Arbor Township and Concord Township. It then flows out of Jackson County and into Calhoun County before it goes through the town of Albion where the North Branch connects with the South Branch and they form to be one river.
According to the U.S. Census Bureau, the county has a total area of 723 square miles (1,870 km2), of which 702 square miles (1,820 km2) is land and 22 square miles (57 km2) (3.0%) is water.[4]
The Jackson County Courthouse was designed by Claire Allen, a prominent southern Michigan architect. Jackson County is also home to the Michigan Whitetail Hall Of Fame.
Jackson County comprises the Jackson, MI Metropolitan Statistical Area.
[1]", named for members of Jackson's Cabinet.Cabinet counties and considered to be one of Michigan's "Andrew Jackson It is named for U.S. President [1]
Battle Creek, Michigan, Barry County, Michigan, Branch County, Michigan, Eaton County, Michigan, Hillsdale County, Michigan
Napoleon Township, Michigan, Norvell Township, Michigan, Parma Township, Michigan, Pulaski Township, Michigan, Rives Township, Michigan
Detroit, Ontario, Ohio, Metro Detroit, Indiana
Ann Arbor, Michigan, Wayne County, Michigan, Monroe County, Michigan, Michigan, Metro Detroit
Michigan, Detroit, Dearborn, Michigan, Metro Detroit, Ann Arbor, Michigan
Huron County, Michigan, Saginaw County, Michigan, Sanilac County, Michigan, Allegan County, Michigan, Newaygo County, Michigan
Northern Michigan, Upper Peninsula of Michigan, Southeast Michigan, Wayne County, Michigan, Central Michigan
Monroe County, Michigan, Washtenaw County, Michigan, Jackson County, Michigan, Hillsdale County, Michigan, Race (United States Census)
Jackson County, Michigan, Michigan, Spring Arbor, Michigan, Jackson, Michigan, Daylight saving time
Jackson County, Michigan, Michigan, Jackson, Michigan, Nascar, Spring Arbor, Michigan


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Rocket Roundup
We would like to welcome our newest "Rockets" to New Haven Community Schools.
Our Roundup events are where you will receive a great deal of information about our Kindergarten and Young 5's programs. This Webpage is designed to give you the information needed to plan to attend these events and also to register your child for next school year. Whether you are checking out what NHCS has to offer or you are ready to get your child signed up for next year, all families are welcome to attend these events.
Thank you for your interest in New Haven Community Schools.
Kindergarten Q & A's


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The Caravelle Resort – 45% Off Cyber Exclusive + Lightning Deals!
Mar 1, 2021 - Jun 1, 2021
- Locations: The Caravelle Resort
Save up to 45% off with our Cyber Sale, the best rates of the year! Featuring no blackout dates and free cancellation (see website for details)! Plus, save BIG on our lightning deals including WonderWorks, Duplin Winery & Board & Brush! For more info call 855-395-7153 or visit our website.
6900 North Ocean Blvd. Myrtle Beach, SC 29572
Located on the famed Golden Mile of Myrtle Beach, The Caravelle Resort offers over 500 units in 9 buildings with a wide variety of rooms, suites & condos. Amenities include an on-site restaurant, oceanfront pool bar, ice cream shop, a variety of pools, whirlpools, lazy river & the Wild Water Pool.


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The most widespread way to reach to millions of people is a web resource. It gives a power to create a unique, absolutely creative image of any organization. Churches fall under the category of institutions, which need a web resource too, because it is a reliable method to attract more believers and members. Ministry websites are different in their visual appeal and technique. Here is a theme with a great combination of awesome design and powerful functionality - dark-colored religious website template with calendar.
The theme has an original coloring – moss green. It is dark though looks rather nice. This web template has a superior laconic design of the home page. The image slider with the top navigation menu looks harmoniously and very effective. Further browsing of this modern church template gives the same feeling of simplicity, style and originality.
24/7 free support
Stock Photos Included
Visual Website Builder


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Mobile, Open Hardware, RISC-V System-on-Chip (SoC) Development Kit
A mind-bending hardware puzzle in three acts
An open source FT2232H-based, multi-protocol, multi-voltage tool for hardware hacking
Voltage glitching like it's 1999
Security keys you create by rolling dice
Support Hack-A-Sat 2020 finalist team ADDVulcan by sporting a slick embroidered patch.
A versatile dev board with everything you need to design custom IoT protocols & gateways
The open source DIY laptop for hacking, customization, and privacy
The One-Chip Hardware Security Module (HSM) and MAX10 FPGA Dev Board
Our third annual party for hacking, discovering, and sharing hardware
A highly capable and extremely flexible open source multitool for digital electronics
A tiny, open source USB computer for security applications
A tiny FIDO2 security key for two-factor authentication and passwordless login
A hardware-based USB 2.0 monitor & trigger platform, controlled from Python
An all-in-one encrypted USB flash drive, two-factor authentication token, and password manager
A handheld RFID & NFC test instrument optimized for untethered use in the field
A Configurable, Affordable System-on-Chip for Analysis and Demonstration of Election Security
Our second annual party for hacking, discovering, and sharing hardware
A 100% libre RISC-V + 3D GPU chip for mobile devices
BadUSB embedded into a USB cable
Open hardware USB true random number generator
A party for hacking, discovering, and sharing hardware
A tool to explore optical data transmissions and covert channels
The world's first RISC-V-based, Linux-capable development board
An open, affordable, compact USB/eSATA-to-Cloud adapter
A low-cost, low-power NAS for 3.5" drives
Physical security for your personal data
A low-cost, low-power NAS for 2.5" drives
An open source USB stick computer for security applications.
Open-source hardware password manager
An open source, physically secure personal computer.
An Earth-friendly way to easily upgrade and fix your own computer
An open hardware security module developed by an independent, international coalition founded to create a trusted, open source, inexpensive, hardware cryptographic engine.


text: Nicolas90 0 Posted October 20, 2016 Hello dear community, I am having trouble scripting a second button on my hda. Both functions for the two buttons have been defined in the OnCreate callback in the scripts tab. While the first one is working fine, clicking the second one throws an error, saying the function or attribute defined in the parameter settings is not available. The callback-parameter for the first button has the value: kwargs["node"].hdaModule().OnCreateCurveButtonPressed() The second one has the parameter: kwargs["node"].hdaModule().OnCreateSectorsButtonPressed() And this is the error message that the console throws: Traceback (most recent call last): File "<stdin>", line 1, in <module> File "C:/PROGRA~1/SIDEEF~1/HOUDIN~1.607/houdini/python2.7libs\hou.py", line 23413, in __getattr__ def _SIDEEF_getattr__(*args): return _hou.HDAModule___getattr__(*args) AttributeError: 'module' object has no attribute 'OnCreateSectorsButtonPressed' Does anybody know whats causing this? Share this post Link to post Share on other sites


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There was good news and bad news for the Kenai. The good news is the sonar counter finally registered a few king salmon. At least three lucky anglers that I know of caught fish. The bad news is every fish caught fell below the 34″ slot limit. Small run, small fish, not a good recipe.
How did I do fishing this week? The string of fishless days continues. I am getting closer to ending the streak, though. I had a bite on Thursday.
The highlight of the fishing week was being able to spend time with my son Mac, his girlfriend Madison, and a few of their friends. Mac and Madison had a busy summer last year in Talkeetna, but now that they're living in Anchorage, I expect to see more of them this summer and fall (hint, hint).
I didn't give the river my full effort this week. With absolutely beautiful weather on the peninsula, Jane said we needed to spend a day or two exploring Homer. With the camper and bikes in tow, we took the hour and a half drive to one of the most scenic towns in Alaska.
Our preferred method to see Homer is on bike. With many bike paths in town, and on the spit, Homer is exceptionally bike friendly.
If you've been to Homer, you know what the photo above is all about. If you've never been to Homer, the Salty Dawg Saloon is one of the most iconic bars in all of Alaska. We seriously thought about stopping in to hydrate, but it was 11 in the morning. We'll save our stop for another day, which is usually after a day of halibut fishing.
Quite a few businesses and restaurants were open in Homer. With many to choose from, this food truck clicked and became our lunch stop. It was a great choice. I would highly recommend A Bus Named Sue to anyone looking for a sandwich or halibut fish and chips.
For the week ahead, I know I'll be back at it again trying to catch a king. It won't be everyday, because Seward is in our sites and this could be the week we check things out there.
See you next week.
COVID UPDATE: Starting June 5th, travelers to Alaska will not have to self quarantine for 14 days if they take a COVID test 72 hours before their flight. This is a good start in getting out state tourists to come to the Great Land.


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Community is the foundation of my platform. I am a firm believer that developing a strong community is integral to building a Cambridge that we are all proud to call home. I have lived in Cambridge my entire life, and in Ward 7 since I was five years old. Growing up I was immersed in our youth sports leagues. I grew up in hockey rinks all year long, I also participated in our youth soccer leagues and softball leagues. These sports were a huge influence on my life and taught me many lessons that I would not have otherwise learned. It is important that we grow and support these parts of our community.
We must provide them with an excellent location for the new recreation facility our city has applied for. We must also support other initiatives that support arts programs, and help build other community connections that get Cambridge citizens out of their houses and enjoying all that this beautiful city has to offer.
The final aspect of community building that I want to focus on is returning Cambridge to the beautiful and safe city that it once was. It is a common theme in talking to people that they no longer feel safe walking in our downtown cores. When people are afraid to walk around downtown it means that any community events that happen will be less successful. It means that businesses have less foot traffic. It means that Cambridge citizens are not coming together to enjoy the beautiful city that we have. This is unacceptable and the problem needs to be addressed. A new location for the Bridges needs to be found and it needs to be outside of the downtown cores.


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Pink Turtle Smocked Day Gown
Begin your sweet baby girl's first layette with our oh so soft, green Turtle daygown with drawstring closure at the bottom. It is the perfect shower or baby gift! Check out the coordinating hooded towel, plush minky, burp cloth, and bib.
- Material: Cotton Pique
- Size: 0-6 months
One size 0-6m
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Think carefully before choosing a laptop with SSD
(MoneyWatch) COMMENTARY The next time you shop for a replacement laptop, you might be offered a new option that didn't really exist a few years ago, the last time you had to get a new PC. Specifically, you might have the option of purchasing a laptop with an SSD -- a solid state drive. But what is an SSD, and do you want one?
The principal behind SSDs is familiar to anyone with a digital camera -- just think about the removable SD or CompactFlash memory card. That memory chip has no moving parts and requires no power to maintain your photos. An SSD is basically the same thing, just not removable, and scaled up to work in a laptop or a PC. It's as if you replaced your hard drive with a memory card.
That might sound good. Common sense dictates that SSDs are faster than hard drives -- no slow, spinning platters -- and more reliable as well -- no failure-prone, spinning platters. But what's the reality? Here are a few things you should know about SSDs before you order a laptops equipped with one.
They're reliable, but not necessarily more so than hard drives. No moving parts -- that almost always means more reliability, right? And indeed, a laptop with an SSD is almost certainly going to survive a 5-foot drop better than a similar laptop with a hard drive. But over the long term, SSDs are proving to have about the same overall reliability as hard drives. For details, you can read the very thorough analysis at Tom's Hardware. Should this scare you away from SSDs? Of course not -- but don't expect them to live forever.
They're expensive. New tech always costs more. SSDs have been around for a number of years, and prices have been dropping steadily. But the reality is that you'll still pay a significant premium for SSD-equipped laptops compared to old-fashioned platters. For example, a typical 256GB SSD drive costs between $180-$250 today. If it could physically fit in a laptop, you'd be able to pack 4TB (that's 4 terabytes) for the same amount of money.
You need a bigger one than you think. And that leads to the final SSD problem -- because they cost so much more, people tend to cut corners and get a smaller SSD to save a few bucks. A couple of years ago, my company bought me a 32GB SSD-equipped laptop, for example. That was the worst PC I'd ever owned. 32GB was barely enough room for Windows and a couple of apps; there wasn't even enough space to install a mission-critical database that I needed to reference every day at work. So how much space do you need? 64GB is the absolute smallest SSD I would consider buying, and I'd actually suggest 128 instead. That is plenty of room for Windows, apps, and all the data you're likely to need.


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Halfway House1 Whitehall Road East
(01274) 680614
Opening times: 12-11
Due to the ongoing coronavirus pandemic this pub is currently offering additional services:
The Halfway House, Birkenshaw, reopened 4th July following the COVID pandemic Orders can be placed on the Halfway House Facebook page then they will ring the customer back with a collection time. Once you arrive they will come out to you, parked in the yellow bay. Growlers, 4 pint capacity, are available for £15 deposit. Cask ale £8 for 4 pints.more information...
Regular beers: Ossett Yorkshire Blonde, Tetley Bitter
See more about this pub on WhatPub, CAMRA's national pub guide.
Busy crossroads traditional ex-Tetley local pub with homely lounge. Monday quiz, Tuesday and Wednesday pool and poker, cash bingo Thursdays, karaoke Friday/Saturday nights, live music of various styles Sundays 6-9. Two guests beers are usually from Yorkshire breweries. Bar snacks, functions catered for in and out.