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Dinko Lukin
Fisherman, problem solver and inventor Dinko Lukin left an indelible mark on global tuna fishing by pioneering the catching and ranching of the legendary Southern Bluefin Tuna with his family-owned company Dinko Tuna. In the 1960s Dinko built the Orao, a boat that became enshrined in Port Lincoln folklore, and first poled tuna in the Great Australian Bight.
In the 1980s government bodies stated overfishing of Southern Bluefin Tuna had put stocks at risk and by 1985 a quota was introduced and enforced for Australian fishermen, with similar restrictions on Japanese and New Zealand fishers. The governing body considered this insufficient and in 1989 tuna fishers had their quota and income cut by two thirds. Dinko, forever the problem solver, threw the industry a lifeline when he hatched the ground-breaking idea of farming this fast swimming ocean fish. In a quest to develop the most sustainable Bluefin tuna fishery in the world, Dinko Tuna implemented the harvest of wild Bluefin Tuna as they pass through the cold, clean waters of South Australia during late summer in February/March.
Using a purse seine method, the live fish are corralled and only a select number are transferred to a net, which is slowly towed back to Port Lincoln where they are ranched. The remaining fish are set free to continue their migration. When the captured fish arrive in Port Lincoln, they are fed a diet of premium, fresh, sustainable pilchards, sardines and yellow tail. The method doubled the fishermen's quota as the limit on their catch was determined by the weight of the fish caught in the wild, with any weight gained in captivity a bonus. Dinko Lukin's solution is now the internationally accepted method for Bluefin Tuna farming.


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Everyone is variable. I thought I was the only variable of the equation of my life. I was wrong, very wrong. People around me move forward, hit the ground, deteriorate, love me, hate me, estrange from me, get close to me, levitate, frustrate, get drunk, become holy, love the devil, and never stop.
At the beginning of this year, I wrote a spontaneous sentence starting with "It could happen…" And one of them seems really happening now. My life can be shifted in the way that I have never projected due to the variable that I mistook as a constant. Ironically, this is the way that I secretly anticipated. Somehow my mind got frozen, vibrating like a large bronze bell hit by an ambitious blow.
Rain, wind, the gloomy sky. Grey, wind, the darkening sky. The blue jay. The geese couple stands so confidently on the road as if they can defeat any foes even the vehicles dashing towards them without slowing. The color of grey-blue forming with many layers calls forth a storm. I feel the wind on my face and my body. Standing still. What is it? What is coming? Did I call it as the clouds in the sky called the rain? Is it coming to me without any notice? The gushing wind warns me. Go inside, go inside. I should. But I stand on the same spot and dare to watch the frowning wind. The deep furrows among the angry clouds. Did I want to be someone? Or something? The big raindrops hit my face. What is coming? What will soak me? What will soak me inside out? Where do all the animals go in this merciless weather? How can they survive when they don't have any inside to go to?
Life. Moves and stops. It usually stops when it isn't expected to stop. Sometimes, it pushes me forward. Or pulls down. Or snaps my hair in the back with a sudden motion. Levity. Give me wings, so I can fly. Make me tumble, make me suffer, make me cry, make me anguish, make me scream with pain, make me pull my hair in the darkest night, make me deceive, make me be deceived, make me fall on my knees, make me lay my head in the cold pit, cover me with giant waves. But don't take my wings. I brush them every night before I sleep. Maybe someday I can fly in my dream. But I only remember the falling after waking up. Then even my dream gets heavy. Heavy. Rain. Wind. Grey sky. Darker nights. Where did the small white butterfly go? My helpless wings.
<May 3rd, 2017>

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- © 2016 American Society of Plant Biologists. All Rights Reserved.
The C4 pathway is a highly complex trait that increases photosynthetic efficiency in more than 60 plant lineages. Although the majority of C4 plants occupy disturbed, arid, and nutrient-poor habitats, some grow in high-nutrient, waterlogged conditions. One such example is Echinochloa glabrescens, which is an aggressive weed of rice paddies. We generated comprehensive transcriptome datasets for C4 E. glabrescens and C3 rice to identify genes associated with adaption to waterlogged, nutrient-replete conditions, but also used the data to better understand how C4 photosynthesis operates in these conditions. Leaves of E. glabrescens exhibited classical Kranz anatomy with lightly lobed mesophyll cells having low chloroplast coverage. As with rice and other hygrophytic C3 species, leaves of E. glabrescens accumulated a chloroplastic phosphoenolpyruvate carboxylase protein, albeit at reduced amounts relative to rice. The arid-grown species Setaria italica (C4) and Brachypodium distachyon (C3) were also found to accumulate chloroplastic phosphoenolpyruvate carboxylase. We identified a molecular signature associated with C4 photosynthesis in nutrient-replete, waterlogged conditions that is highly similar to those previously reported from C4 plants that grow in more arid conditions. We also identified a cohort of genes that have been subjected to a selective sweep associated with growth in paddy conditions. Overall, this approach highlights the value of using wild species such as weeds to identify adaptions to specific conditions associated with high-yielding crops in agriculture.
All photosynthetic organisms rely on the primary carboxylase activity of Rubisco to produce carbohydrates for life. However, since Rubisco evolved with a limited capacity to distinguish between CO2 and oxygen (Sage, 2004), rates of oxygenation can have a significant impact on photosynthetic efficiency (Ehleringer and Monson, 1993). In response to evolutionary pressures, approximately 30 million years ago a complex phenotype known as C4 photosynthesis evolved in more than 60 plant lineages (Sage et al., 2011, 2012). C4 photosynthesis is a carbon-concentrating mechanism that allows Rubisco to operate in a CO2-enriched environment, thus avoiding the problems of oxygenation. Phosphoenolpyruvate carboxylase (PEPC), a cytosolic enzyme that does not react with oxygen and has a higher affinity for carbon than Rubisco, performs the initial carboxylation reaction (Hatch, 1987). The resulting C4 acids are subsequently decarboxylated in the vicinity of compartmentalized, chloroplastic Rubisco. At least 25 anatomical types capable of achieving the necessary compartmentation for C4 photosynthesis have been described (Edwards and Voznesenskaya, 2011). In most C4 species, specialized cell-types within the leaf are arranged in "Kranz" formation (Sage, 2004). Veins are surrounded by concentric rings of bundle sheath (BS) and then mesophyll (M) cells, with PEPC and Rubisco operating in M and BS cells, respectively, and organic acids moving between them.
The C4 biochemical cycle also varies between lineages of C4 plants and has commonly been categorized into three subtypes (Fig. 1). These biochemical subtypes are based on whether NADP-dependent malic enzyme (NADP-ME), NAD-dependent malic enzyme (NAD-ME), or phosphoenolpyruvate carboxykinase (PEPCK) releases CO2 from the C4 acids. However, it is now recognized that mixed subtypes utilizing multiple C4 acid decarboxylases are common and provide selective advantage (Furbank, 2011; Bellasio and Griffiths, 2014; Wang et al., 2014a). Maize (Zea mays), for example, uses both NADP-ME and PEPCK to release CO2 in BS cells (Hatch, 1971; Chapman and Hatch, 1981; Furumoto et al., 1999; Wingler et al., 1999; Furumoto et al., 2000; Majeran et al., 2010; Pick et al., 2011). Models suggest this flexibility helps maintain energy balances and allows for reduced diffusion requirements of acids between M and BS cells in a fluctuating environment (Wang et al., 2014a).
The C4 carbon-concentrating mechanism has a wide-ranging impact on plant physiology. Due to the high affinity of PEPC for carbon, C4 leaves maintain lower stomatal conductance than C3 leaves, and have lower rates of water loss per unit of carbon fixed and higher water-use efficiencies (Taylor et al., 2010). Photosynthetic efficiency per unit nitrogen is also increased in C4 species (Long, 1999) because the enriched CO2 environment surrounding Rubisco allows for a reduction in the amount of Rubisco for the Calvin-Benson cycle. These increases in water- and nitrogen-use efficiencies are thought to contribute to the preponderance of C4 species in relatively dry environments that can also be nutrient poor and disturbed (Osborne and Sack, 2012; Sage et al., 2012). However, the C4 trait is broadly adaptive, with, for example, some C4 species thriving in nutrient-rich, waterlogged conditions. In fact, the most productive native species on the planet, Echinochloa polystachya, is an aquatic C4 grass that occupies monospecific stands in the nutrient-rich Amazonian basin (Piedade et al., 1991). There is little information on photosynthesis in these highly productive aquatic C4 species.
Other C4 Echinochloa species are also highly productive in waterlogged conditions and some of the worst weeds in the world. One such species is Echinochloa glabrescens (barnyard grass), which is an aggressive weed of paddy rice (Fig. 2A). Echinochloa species have operated the C4 pathway in waterlogged conditions for a significant period of time. Archaeological evidence documents the occurrence of Echinochloa species in rice paddies 8,000 years ago (Gross and Zhao, 2014), and although E. glabrescens is phylogenetically distant to rice (Fig. 2B), selective forces imposed over time from hand weeding are thought to have led to it becoming a rice biomimic (Barrett, 1983). E. glabrescens therefore provides an opportunity to investigate both how C4 photosynthesis operates in waterlogged, nutrient-replete conditions generally and in the rice paddy specifically. It also allows for an investigation of genetic adaptation to paddy growth conditions irrespective of the photosynthetic state of a species.
In this study, we define the anatomical arrangement of M and BS cells in E. glabrescens and the biochemical subtype associated with release of CO2 in the BS. In addition, two M characteristics previously described in rice were investigated to better understand whether they are general adaptations to aquatic growth or are species specific. First, we assessed the degree of M-cell-lobing and chloroplast coverage in E. glabrescens to determine whether biomimicry extends to cellular ultrastructure. Rice M cells are heavily lobed with chloroplasts lining the entire cell periphery (Sage and Sage, 2009). Second, we investigated whether E. glabrescens possesses a chloroplast-localized PEPC. Rice uses a chloroplastic PEPC to balance carbon and nitrogen metabolism in paddy conditions where most nitrogen is supplied as ammonium (Masumoto et al., 2010), but it is not known whether C4 photosynthesis can operate in the presence of a chloroplastic PEPC. In addition to addressing these specific questions, we generated comprehensive transcriptome data for E. glabrescens to provide unbiased insight into its photosynthetic characteristics specifically and patterns of gene expression more generally. Comparison with deep sequencing data from C3 rice and C4 maize identified repeated alterations to patterns of gene expression associated with the evolution of life in waterlogged conditions or the complex C4 phenotype.
Anatomical and Physiological Traits of E. glabrescens
To investigate Kranz anatomy and lobing in C4 E. glabrescens, transverse and tangential sections of E. glabrescens were generated. In cross section, E. glabrescens leaves exhibited classical Panicoid-type C4 anatomy with M cells surrounding BS cells in concentric circles around the vasculature (Fig. 3). M cells were minimally lobed in both cross (Fig. 3A) and tangential (Fig. 3B) sections, and no lobing was apparent on the BS. On average, M and BS cells were similar in size (Fig. 3D). However, chloroplast area in BS cells was approximately twice that found in M cells on both a cell area and percentage of cell area basis (Fig. 3D). M chloroplasts were distributed close to the cell walls and contained extensive granal stacking (Fig. 3C). BS chloroplasts were centrifugally distributed, accumulated starch, and contained mostly thylakoids with little to no granal stacking (Fig. 3C).
We next quantified photosynthetic characteristics of both species (Fig. 4). The presence of PEPC in leaves of C4 species leads to lower carbon isotope discrimination (von Caemmerer et al., 2014) and lower compensation points (Sage, 2004). Photosynthesis in C4 species also saturates at higher light levels than in C3 plants (Ehleringer and Monson, 1993). Carbon isotope discrimination, photosynthetic assimilation rates under the conditions of growth (Anet), CO2 compensation points, carboxylation efficiencies, maximum photosynthetic rates (Amax), and assimilation rate of E. glabrescens and rice were consistent with C4 and C3 photosynthesis, respectively (Fig. 4E). Anet and the carboxylation efficiency of C4 E. glabrescens were approximately 2.7 and 13 times higher than in C3 rice, respectively. These data, taken together with the structural traits, confirm that E. glabrescens was running an effective C4 cycle. We next sought to better understand the patterns of gene expression associated with C4 photosynthesis in E. glabrescens and also those genes associated with waterlogged conditions in the paddy environment.
De Novo Assembly and Annotation of the E. glabrescens Transcriptome
Flow cytometric analysis was used to determine that the size of the E. glabrescens genome is approximately 14 times larger than that of Arabidopsis thaliana and approaching the size of the maize genome (Table I). The E. glabrescens genome was approximately twice the size of other species in its genus (Zonneveld et al., 2005), some of which have previously been reported to be allohexaploids (Hilu, 1994; Ye et al., 2014). As there is neither a reference genome for E. glabrescens nor a genome from a closely related species that can be used for scaffolding, we used de novo assembly followed by contig annotation to define the gene complement and then quantify gene expression in this species (Aubry et al., 2014).
Sequencing of RNA from E. glabrescens and rice resulted in 74 million and 70 million reads, respectively (Table II). De novo assembly of E. glabrescens generated 50,226 contigs, 31,151 of which could be annotated to 15,212 orthologous genes in rice (Table II). A total of 15,147 orthologous transcript clusters had nonzero expression in at least one sample after quantification and were used in subsequent analyses. Spearman's rank correlation of transcript abundance indicated good agreement between biological replicates (Supplemental Fig. S1A).
Global Analysis of Transcript Abundance in E. glabrescens and Rice
To investigate major differences in transcript accumulation between rice and E. glabrescens, 15,147 orthologous transcript clusters were categorized into four possible statistical patterns consisting of those that showed equal expression in both species (posterior probability of differential expression [PPDE] ≤ 0.01), those that were more or less abundant in E. glabrescens relative to rice (both at PPDE ≥ 0.99), and those with no significant pattern (0.01 < PPDE < 0.99; Fig. 5A; Supplemental Table S1). Genes equally expressed in both species were included as a category to identify genes likely to be involved in fundamental processes not affected by photosynthetic type.
To obtain an overview of alterations in transcript abundance between C3 rice and C4 E. glabrescens gene ontology (GO) terms were used. Overall, 152 GO terms were statistically overrepresented across all four transcript patterns (P < 0.05). Transcripts that were more or less abundant in E. glabrescens compared with rice accounted for 74% of the overrepresented terms (Supplemental Table S2). GO terms encapsulating electron carrier activity (GO:0009055), thylakoids (GO:0009579), and oxidoreductase activity acting on NAD(P)H, quinone or similar acceptors (GO:0016655), were overrepresented in the genes up-regulated in C4 E. glabrescens relative to rice. Genes more abundant in rice were associated with GO terms for ribosomes (GO:0003735, GO:0005840) and translation (GO:0008135, GO:0006412), reflecting the relatively large investment in Rubisco, Calvin-Benson cycle, and photorespiratory pathway necessary in C3 species (Bräutigam et al., 2011). GO terms related to basic processes such as the cell-cycle (GO:0007049), histone-binding (GO:0042393), and multicellular organismal development (GO:0007275) were associated with transcripts that had equal expression between species.
To provide insight into differences in regulators of gene expression in leaves of rice and E. glabrescens, we investigated expression of transcription factors (TF) in each species. 1092 TFs distributed across 87 classes were detected, of which 891 were significantly differentially expressed between C3 rice and C4 E. glabrescens or equally expressed in both species and potentially involved in basic metabolism. A previous comparison of developing leaves of C3 rice and C4 maize led to the identification of 118 TFs proposed to play a role in regulating C4 photosynthesis (Wang et al., 2014b), and 91 of these TFs were differentially expressed in our dataset (Supplemental Table S3). Of these, 31 were more abundant in C4 E. glabrescens than in C3 rice, and analysis of publicly available datasets showed that 90% of these TFs had cell-enriched expression patterns in maize or Setaria viridis (Li et al., 2010; Chang et al., 2012; John et al., 2014; Wang et al., 2014b). 34 TFs were less abundant in C4 E. glabrescens than C3 rice, but 82% of these also exhibited cell-enriched expression in M or BS cells of S. viridis or maize (John et al., 2014; Wang et al., 2014b). Quantitative PCR (Q-PCR) analysis of mRNA isolated from M and BS cells of E. glabrescens showed that seven of the eight transcription factors assessed were preferentially expressed in either cell type (Supplemental Fig. S2), although not always in the same cell-type as has been found in S. viridis and maize. This finding indicates that these separate C4 lineages up-regulate homologous transcription factors, but interestingly not always in the same cell-type.
C4 Cycle Proteins and Related Isoforms in E. glabrescens
To investigate the C4 cycle in E. glabrescens, the transcriptome data were mined for homologs to known C4 proteins. As expected, transcripts related to the core C4 cycle accumulated at higher levels in E. glabrescens than rice, and multiple isoforms were detected for those genes that are part of multigene families (Supplemental Table S4).
Five homologs of the rice PEPC gene family were detected in the E. glabrescens transcriptome. To investigate whether any of these transcripts encoded a chloroplast targeted protein, the predicted amino acid sequences were aligned against the rice chloroplastic PEPC (Os01g11054). E. glabrescens transcript_8661 possessed an N-terminal extension, and the predicted amino acid sequence did not contain an Ala to Ser substitution highly correlated with the C4 enzymatic isoform of PEPC (Bläsing et al., 2000; Christin et al., 2007). Transcript_8661 was approximately 4-fold less abundant in C4 E. glabrescens than C3 rice and 286 times less abundant than the most highly expressed PEPC in E. glabrescens (Supplemental Table S4). A translational fusion between the full coding sequence of transcript_8661 and GFP was found to be chloroplast targeted when tested for subcellular localization in planta (Fig. 6). To investigate whether chloroplastic PEPC is only present in hygrophytic species (Masumoto et al., 2010; Fukayama et al., 2014), publicly available databases were searched for PEPC proteins with an N-terminal extension. Fifteen annotated sequences in addition to the rice protein were detected. ChloroP suggested that PEPC proteins from C4 Setaria italica and C3 Brachypodium distachyon were chloroplast targeted (Emanuelsson et al., 1999; Supplemental Table S5). Translational fusions of the first 55 residues of Si000160m and the first 48 residues of Bradi2g06620 to GFP were sufficient for chloroplast localization (Fig. 6). It therefore appears that chloroplastic PEPCs are not restricted to species adapted to waterlogged conditions and are found in multiple C3 (rice, B. distachyon) and C4 (E. glabrescens, S. italica) species (Fig. 6).
Transcripts for multiple isoforms of the C4 decarboxylase enzymes NADP-ME and NAD-ME and a single isoform of PEPCK were also detected (Supplemental Table S4). While NAD-ME encoding transcripts were either up in rice relative to E. glabrescens or had no significant pattern of expression, NADP-ME and PEPCK encoding transcripts were significantly more abundant in E. glabrescens. In particular, E. glabrescens transcripts most similar to Os01g09320 (NADP-ME) and Os03g15050 (PEPCK) were 43 and 124 times more abundant in E. glabrescens relative to rice, respectively. Immunoblotting confirmed that the NADP-ME, PEPCK, and other C4 cycle enzymes were abundant in E. glabrescens (Fig. 7). PEPC and pyruvate, orthophosphate dikinase (PPDK), which had transcripts 423 and 26 times more abundant in E. glabrescens than in rice, were also more abundant in E. glabrescens at the protein level. In contrast, carbonic anhydrase (CA) protein was more abundant in rice, while transcripts for two detected CA genes were up-regulated in E. glabrescens (Supplemental Table S4). Malate dehydrogenase (MDH) encoding transcripts were nearly 10 times more abundant in E. glabrescens but did not differ dramatically between species at the protein level (Supplemental Table S4; Fig. 7).
Transcripts encoding metabolite transporters associated with the C4 pathway were more abundant in E. glabrescens than in C3 rice, with the exception of DICARBOXYLATE TRANSPORTER2 (DCT2), which had no significant pattern of expression (Supplemental Table S4). The transporters analyzed are known to be more abundant in maize relative to C3 Pisum sativum (Bräutigam et al., 2008) and enriched in M or BS cells of maize (Taniguchi et al., 2004; Majeran et al., 2005; Majeran et al., 2008; Majeran et al., 2010). Consistent with the lower amounts of Rubisco in C4 leaves and reduced rates of photorespiration, RBCS transcripts (encoding the small subunit of Rubisco) as well as transcripts related to photorespiration were reduced in E. glabrescens compared with rice (Supplemental Table S4). Overall, we conclude from the transcript and protein data that the aquatic C4 species E. glabrescens is operating a C4 pathway that relies on both NADP-ME and PEPCK to provide CO2 to Rubisco in the BS. The data also indicate that a chloroplastic PEPC in C4 leaves is compatible with operation of the C4 cycle, although in C4 E. glabrescens transcripts encoding the chloroplastic PEPC are down-regulated relative to C3 rice (Supplemental Table S4).
Genes Associated with Paddy Conditions and C4 Photosynthesis
To provide broader insight into genes associated with either the paddy environment or C4 photosynthesis, we next included publicly available data for the C4 grass maize grown in nonpaddy conditions in our analysis (Wang et al., 2013a). These data allowed a three-way comparison consisting of two species that evolved the C4 pathway independently (E. glabrescens and maize; Giussani et al., 2001; Christin et al., 2008; Sage et al., 2011), and also two species that independently adapted to life in waterlogged paddy conditions (E. glabrescens and rice). Transcript abundance was highly correlated between biological replicates of each species (Supplemental Fig. S1B), but maize and E. glabrescens, both members of the PACMAD clade (Fig. 2), showed higher correlations with each other than with rice.
In total, 12,256 rice loci were detected in all three species and analyzed for statistical differences in transcript accumulation according to eight possible patterns of expression (Fig. 5, B and C; Supplemental Table S6): up in both C4 species relative to C3 rice, up in rice relative to the C4 species (equivalent to down in C4), up in both paddy species relative to maize, up in maize relative to the paddy species (equivalent to down in paddy), up and down in E. glabrescens relative to both rice and maize (representing this species' unique expression profile), equal expression between species (representing genes involved in basic metabolism), and no significant pattern between species. While nearly half the genes had no significant pattern of expression (Fig. 5B), putative paddy- or C4-related genes constituted 18% and 15% of the detected rice loci, respectively, and 16% of the genes corresponded to an expression profile that was unique to E. glabrescens. Approximately 5% of genes were equally expressed between species and likely represent basic metabolism.
E. glabrescens and maize had similar, but not identical, molecular signatures for the core C4 pathway, metabolite transport, and photorespiratory process (Supplemental Tables S4 and S6). For the core C4 pathway genes, with the exception of CA, the gene family member having the most abundant expression in E. glabrescens relative to rice in the two-species analysis was also up-regulated in both C4 species in the three-species analysis. Similarly, at least one isoform of the transporters PHOSPHOENOLPYRUVATE/ PHOSPHATE TRANSLOCATOR (PPT), DICARBOXYLATE TRANSPORTER1 (DCT1), METHYL ERYTHRITOL PHOSPHATE2 (MEP2), and TRIOSE PHOSPHATE/PHOSPHATE ANTIPORTER (TPT) was up-regulated in both C4 species. However, the CA and MEP3b homologs most abundant in E. glabrescens relative to rice were up-regulated in the paddy species relative to maize, DCT2 was down-regulated in the paddy species relative to maize, and no significant pattern of expression was found for 2-OXOGLUTARATE/ MALATE TRANSPORTER (OMT1) and MEP3a. Although the photorespiratory genes were not statistically classified as up in rice relative to the C4 species (i.e. down in C4), transcript accumulation was typically at least an order of magnitude higher for one isoform per gene in rice relative to the C4 species (Supplemental Tables S4 and S6), consistent with a more active photorespiratory pathway in C3 rice.
Fifty-five GO terms were statistically overrepresented across the eight expression categories, of which 17 and 12 were associated with genes up-regulated in paddy or C4 species, respectively (Supplemental Table S7). Terms related to the nonoxidative branch of the pentose phosphate pathway and/or the Calvin-Benson cycle were associated with genes up-regulated in the paddy (GO:0009052, GO:0004751), possibly indicating a stronger repression of these pathways in maize with respect to rice and E. glabrescens. With respect to C4, two GO terms annotated as ATP proton transport (GO:0015991, GO:0046961) were associated with up-regulated genes. This is consistent with the higher demand for ATP generated by the C4 cycle relative to C3 biochemistry. The Glu biosynthetic process (GO:0006537) was also associated with the C4 species, providing further support for an operational PEPCK C4 cycle in E. glabrescens.
TFs were analyzed to gain insight into regulatory networks that might be operating on the C4- or paddy-related gene sets. 967 TFs belonging to 86 families were identified, with 117 and 132 TFs more abundant in the C4 and paddy species, respectively. To determine whether the C4- or paddy-associated TFs might be coregulated, a motif search of the first 1000 bp of the rice homologs for each set of TFs using ELEMENTS was performed (Mockler et al., 2007). Seven and 16 motif clusters, up in C4 and up in paddy respectively, were found (P < 0.05; Supplemental Table S8). The TFs identified in the three-species analysis were also compared with the 118 TFs previously proposed to be associated with C4 photosynthesis (Wang et al., 2014b). The same 91 TFs present in the rice vs. E. glabrescens analysis were distributed across five of the patterns of expression present in the three-species analysis (Supplemental Table S9), but only 12 TFs were classified as up in both C4 species. These 12 TFs were also up-regulated in E. glabrescens in the two-species analysis, suggesting they may play a strong role in C4 regulation. Indeed, the rice homolog of SCARECROW, a BS-enriched gene proposed to be involved Kranz anatomy development (Slewinski et al., 2012; Slewinski, 2013; Wang et al., 2013a), was part of this list. It was also notable that 9 of 12 TFs up in the C4 species have BS-enriched expression in the tip of maize leaves (Li et al., 2010), and when a motif search was performed using ELEMENT, an exact match for a candidate BS cell cis-element from Wang et al. (2014b) was found (Supplemental Tables S10 and S11), suggesting that a egulatory network present in C3 rice continues to function in maize and E. glabrescens but in a cell-enriched manner. It is also notable that 14 of the TFs previously identified as having a role in C4 photosynthesis (Wang et al., 2014b) were up in both paddy species and that they were roughly split between BS- and M-enriched expression in maize (Li et al., 2010). Motif analysis using ELEMENT (Supplemental Table S10) yielded three candidate cis-elements that were present in both the Wang et al. (2014b) dataset and the TFs up-regulated in the paddy species (Supplemental Table S11).
The GLK TF family is required in plants for chloroplast development and photosynthetic gene expression (Hall et al., 1998; Rossini et al., 2001; Fitter et al., 2002; Waters et al., 2009; Powell et al., 2012; Wang et al., 2013b; Nguyen et al., 2014) and also plays a role in regulating nitrogen metabolism in Arabidopsis by inducing Gln synthetase expression and repressing Glu dehydrogenase (Gutiérrez et al., 2008). GLKs are usually present as a duplicated gene pair whose members are subfunctionalized in C4 species between M and BS cells but operate redundantly in all photosynthetic cells of C3 plants (Wang et al., 2013b). A GLK gene pair previously identified as OsGLK1 (Os06g24070) and OsGLK2 (Os01g13740; Wang et al., 2013b) was up in both paddy species in the three-species analysis and up in rice relative to E. glabrescens in the two-species analysis. A second related gene pair, Os04g56990 and Os05g40960, was up-regulated in E. glabrescens relative to rice in the two-species analysis, with Os04g56990 up in both C4 species and Os05g40960 having no significant pattern in the three-species analysis. These two pairs of genes related to GLK may reflect subfunctionalization needed to perform the dual roles of chloroplast development and nitrogen assimilation in both paddy (Pair 1: Os06g24070, Os01g13740) and C4 (Pair 2: Os04g56990, Os05g40960) contexts. Homologs of Os06g24070 and Os04g56990 are M-enriched and Os01g13740 and Os05g40960 are BS-enriched in maize (Li et al., 2010).
Transcripts Up-Regulated in Paddy Conditions Are Evolving More Rapidly
The analysis above identified 1610 transcripts putatively important to C4 function in both dry and wet conditions and also 1905 genes with a potential role in paddy life. To investigate whether these groups of genes have been evolving at different rates compared with the general population of genes, we assessed the number of nonsynonymous substitutions per site (Fig. 8). Interestingly, genes up-regulated in the paddy species have been evolving approximately 10% faster than those that are either down-regulated or nondifferentially expressed (P = 0.004; Fig. 8). This suggests that genes up-regulated in the two paddy species may have evolved to adapt to waterlogged conditions. An analogous effect is not seen for genes that are up- or down-regulated in C4 species compared to C3. However, we note that genes that are up-regulated in C4 species may be evolving faster (P = 0.018) than those that are not significantly differently expressed under any other condition.
Photosynthetic Characteristics of E. glabrescens
M cells of E. glabrescens were similar to arid C4 species, having minimal lobing as well as low chloroplast coverage of the cell area and periphery (Fig. 3; Panteris and Galatis, 2005; Stata et al., 2014). As such, although E. glabrescens seedlings are highly effective at mimicking rice leaf architecture at a gross level, M cell ultrastructure was similar to C4 maize (Panteris and Galatis, 2005) rather than rice. In addition, M chloroplasts of E. glabrescens were distributed along the cell periphery and, as with other C4 species, are partially recessed from the cell wall to create a cytoplasmic channel where PEPC can fix carbon (Stata et al., 2014). This is unlike both domesticated and wild rice species, which exhibit extensive M-cell-lobing with chloroplasts and stromules covering over 97% of the cell periphery (Sage and Sage, 2009). It also contrasts with C3 and C2 (C3-C4 intermediate) species, which have more M chloroplasts than C4 species and a high degree of appression against the cell wall (Stata et al., 2014). Placement of M cell chloroplasts against the cell wall is thought to act as a partial photorespiratory CO2-scavenging mechanism by forcing CO2 to pass through chloroplasts when diffusing both into and out of the cell (Sage and Sage, 2009; Stata et al., 2014). Such an arrangement would be unnecessary in C4 M cells since PEPC acts as carboxylase in the cytosol and Rubisco operates in the BS (Stata et al., 2014). Physiological characteristics were all in the C4 range, and gas exchange analysis of E. glabrescens showed high rates of photosynthesis relative to rice (Fig. 4). Taken together, the anatomical and physiological features of E. glabrescens were those of a classical Panicoid C4 species similar to other arid C4 grass species.
Chloroplast-Localized PEPC
Our data suggest a chloroplastic PEPC that provides carbon skeleton for ammonium assimilation is important to paddy life for both C3 and C4 species. Chloroplastic PEPC is present in two monocot C3 hygrophytic species, rice (Masumoto et al., 2010) and Monochoria vaginalis (water hyacinth; Fukayama et al., 2014), as well as C4 E. glabrescens (Fig. 6). In these paddy species, chloroplastic PEPC is likely an adaptation to the reductive environment of the waterlogged growth habitat where all inorganic nitrogen is available as NH4+ (Masumoto et al., 2010). However, the presence of a chloroplast-localized PEPC in both the arid-growing C3 weed B. distachyon and the cultivated C4 S. italica suggests that either dry environments also lead to the evolution of a chloroplastic PEPC functioning in ammonium metabolism, or that this enzyme has undiscovered roles. The rapidly growing list of species with published transcriptomes can be used to explore the presence and purpose of chloroplast-targeted PEPC across a broad phylogenetic context.
It was previously uncertain whether chloroplastic PEPC in rice would complicate engineering efforts to introduce C4 photosynthesis into rice in order to increase yields (Kajala et al., 2011). Although transcripts encoding chloroplastic PEPC were less abundant in E. glabrescens than in rice (Supplemental Table S4), chloroplastic PEPC localization in multiple highly successful C4 species (S. italica and E. glabrescens; Fig. 6) suggests its activity is compatible with the C4 cycle. This is likely aided by spatial restriction of most PEPC accumulation to M cells where both cytosolic and chloroplastic PEPCs would positively contribute to C4 shuttling (Fig. 9). In addition, any PEPC activity in C4 BS cells, if expression occurs in these cells, would likely be limited to non-C4 related pathways because PPDK has M-enriched accumulation in C4 species. Of further benefit to engineering efforts, rice chloroplastic PEPC appears to behave very differently from the cytosolic isoform (Fukayama et al., 2006; Masumoto et al., 2010; Fukayama et al., 2014; Muramatsu et al., 2015). Relative to cytosolic PEPC, the rice chloroplastic PEPC has very low affinity for PEP; low sensitivity to the inhibitors Asp, Glu, and malate; and it is activated by Glc-1-phosphate, Fru-1,6-bisphosphate, 3-phosphoglyceric acid, and 2- phosphoglyceric acid, but not by Glc-6-phosphate or alkaline pH (Muramatsu et al., 2015). Cytosolic and chloroplastic PEPCs are also nocturnally and likely diurnally regulated respectively (Fukayama et al., 2006; Fukayama et al., 2014). As such, it is possible that when cytosolic and chloroplastic PEPCs are both present in C4 species, they are active at different times of day and have different enzymatic properties, allowing for balancing of C4 and nitrogen metabolism. Activity assays and temporal expression analyses of the chloroplast targeted PEPC gene in the C4 species E. glabrescens and S. italica are required to better understand how the operation of these two PEPCs is integrated.
Balancing Carbon and Ammonium Metabolism in a C4 Paddy Species
A mixed NADP-ME/PEPCK C4 cycle may play an important role in linking carbon and nitrogen metabolism in E. glabrescens and in maintaining adequate C4 flux in a paddy environment (Fig. 9). Presence of a mixed NADP-ME/PEPCK cycle is supported by centrifugally positioned BS cell chloroplasts with reduced granal stacking (Gutierrez et al., 1974; Nelson and Dengler, 1992; Fig. 3), high protein abundance of NADP-ME and PEPCK (Fig. 7), and high transcript levels of NADP-ME, PEPCK, ASP AMINOTRANSFERASE (Asp-AT) and ALA AMINOTRANSFERASE (Ala-AT) relative to both maize and rice (Supplemental Table S6). In addition, E. glabrescens possesses a highly abundant chloroplastic CA (Os01g45274) that could provide bicarbonate to the chloroplastic PEPC. Transcripts encoding this CA isoform was approximately 9-fold and 2-fold more abundant than in maize and rice, respectively (Supplemental Table S6), and the corresponding E. glabrescens transcript_4427 only differed by one residue to the peptide sequence of Os01g45274, which is known to be chloroplast-localized (Suzuki and Burnell, 1995).
A mixed NADP-ME/PEPCK cycle would be able to provide PEP to carbon and nitrogen metabolism through a number of routes (Fig. 9). Both cytosolic and chloroplastic PEPCs in the M could be directly provided with PEP by PEPCK activity in BS cells. In support of this notion, PEPCK (Os03g15050) transcripts are approximately 4-fold and 129-fold more abundant in E. glabrescens relative to maize and rice, respectively (Supplemental Table S6). In addition, PEP could be generated from pyruvate in M cell chloroplasts via PPDK operating in the NADP-ME C4 cycle. Pyruvate could be transferred from BS to M cells either directly or via Ala. E. glabrescens transcripts most similar to NADP-ME genes Os01g09320 and Os05g09440 are approximately 4-fold and 8-fold more abundant than in maize and 45-fold and 4-fold more abundant than in rice (Supplemental Table S6), and transcripts encoding ADENYLATE KINASE (AK) (Os08g01770), which drives the PPDK reaction toward PEP (Burnell, 2011), accumulate approximately eight times and 11 times higher in E. glabrescens relative to maize and rice (Supplemental Table S6). Since maize also operates a mixed NADP-ME/PEPCK cycle (Hatch, 1971; Chapman and Hatch, 1981; Furumoto et al., 1999; Wingler et al., 1999; Furumoto et al., 2000; Majeran et al., 2010; Pick et al., 2011), the abundant accumulation of NADP-ME and AK transcripts in E. glabrescens suggests that increased pyruvate and PEP in M cells is both possible and necessary for the operation of C4 and ammonium metabolism.
To support sufficient levels of metabolite flux for both C4 and ammonium metabolism, abundant expression of transporters might be expected in E. glabrescens. Indeed, transcripts for PPT (PEP transporter), TPT (3-phosphoglycerate/triosephosphate exchanger), DCT1 (Glu/malate exchanger), MEP2 (putative OAA/malate shuttle), and OMT1 (2-oxoglutarate/malate exchanger) are all more abundant in E. glabrescens relative to maize and rice. Of particular note, MEP3b, a putative pyruvate transporter, is 40 times more abundant in E. glabrescens than in maize. Although the Arabidopsis homolog of MEP3b acts as a glycolate glycerate transporter (Pick et al., 2013), the role of MEP proteins in maize has, to our knowledge, not yet been defined. Overall, the data imply that E. glabrescens likely has sufficient flexibility to provide flux to both C4 and ammonium metabolism as needed and so is able to maintain high photosynthetic rates (Fig. 4).
E. glabrescens Transcriptome Yields C4- and Paddy-Associated Genes
Differential expression analysis of rice (paddy C3), E. glabrescens (paddy C4), and maize (arid C4) identified 1610 genes, including 117 TFs putatively important to C4 function. E. glabrescens and maize are descended from two independent lineages of C4 evolution (Giussani et al., 2001; Christin et al., 2008), but both are members of the same PACMAD clade (Fig. 2). As a result, it is presently unclear whether the up-regulation of those genes in the two C4 species relative to rice is due to independent recruitment to the C4 pathway or phylogenetic signature. In either case, it is interesting to note that 91 of the TFs have previously been characterized as putative C4 regulators (Wang et al., 2014b), but only 12 of those TFs were up-regulated in both E. glabrescens and maize (Supplemental Table S9). These 12 TFs form key targets for C4 functional analyses since they exhibit similar behavioral patterns across at least two C4 species. In addition, E. glabrescens and maize had unique molecular signatures with 3% and 2% of detected genes respectively up-regulated solely in one species. Some of these genes may be specifically required for either wet or dry C4 photosynthesis, and future comparative studies should consider further wet C4 species to address this. Interestingly, the majority of core C4 genes and transporters appear to have similar expression profiles, with the exception of genes playing a dual role in ammonium metabolism, suggesting genes associated with recruitment to C4 photosynthesis can dynamically interface across multiple pathways.
A putatively paddy-related signature of 1905 genes was also identified in the three-species analysis. This gene suite appears to be evolving more rapidly relative to genes that were nondifferentially expressed or down-regulated in paddy (Fig. 8) and so can be used to identify essential paddy genes by comparative expression analysis with rice varieties adapted to dry upland growth conditions and/or other paddy weeds. Major alterations in transcript abundance associated with life in paddy conditions could be mediated by convergent alterations to elements in cis in each genome, changes to transfactors, or a combination of both. Without E. glabrescens genome information, it is not possible to comprehensively investigate alterations in cis, but the 132 TFs identified as under selection constitute a manageable starting point for characterizing genes of possible benefit to rice-breeding programs. As rice agriculture must rapidly adjust to changes in climate that affect paddy conditions, e.g. using reduced water levels, these TFs and other genes identified as being under selection in the paddy species could be mined for diversity that would help improve rice stocks.
In summary, the analysis presented provides, to our knowledge, the first insight into leaf transcript populations of a species performing C4 photosynthesis in wet paddy conditions. The data indicate that E. glabrescens runs a mixed C4 biochemical pathway dependent on both chloroplastic NADP-ME and cytosolic PEPCK decarboxylases. We propose that a chloroplastic PEPC combined with abundant PEPCK allows for efficient metabolic flux through both C4 and ammonium metabolism in E. glabrescens. In addition, comparative expression analysis between the three species E. glabrescens, maize, and rice has laid the foundation for identifying a molecular signature for paddy life.
Echinochloa glabrescens was grown from seed in a University of Toronto greenhouse in 20 L pots of sandy-loam. The approximate day/night temperature of the greenhouse was 27/26°C, and peak midday irradiance exceeded 1600 μmol photons m−2 s−1. Fertilizer was supplied as a 50:50 mixture of Miracle-Gro 24-10-10 All Purpose Plant Food and Miracle Gro Evergreen Food (30-110-20) at the recommended dosage (22 mL of fertilizer salt per 6 L; Scotts Micracle-Gro). Plants were sampled between 10:00 and 11:00 AM. Internal leaf anatomy was assessed on sections harvested from the middle of the leaf (1 leaf per plant; 3 plants) and prepared for light and transmission electron microscopy as described previously (Sage and Williams, 1995). Quantification of transmission electron microscopy (TEM) images was prepared from three leaf sections from three separate plants. TEM images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA) to quantify cell area (n = 11), chloroplast area per cell (M n = 11; BS n = 10), and the percent chloroplast area per cell (M n = 11; BS n = 10). Cross sections of cleared E. glabrescens leaf tissue from plants grown at the University of Cambridge (see Physiology below, same seed stock) were used to calculate vein density. Tissue was cleared of chlorophyll by immersion in 70% ethanol at 65°C for 24 h followed by 5% w/v NaOH at room temperature for 24 h. Samples were washed three times in distilled water before examination. Images were taken on a light microscope using Leica Application Suite (Leica, Germany) at 4× magnification and analyzed using ImageJ (National Institutes of Health, Bethesda, MD, USA).
Seeds of E. glabrescens and Oryza sativa L. ssp. indica cv IR64 were germinated at the University of Cambridge in a growth chamber at 28°C, 50% humidity, 400 ppm CO2, and 400 µmol photons m−2 s−1 light in a 16 h photoperiod. Seedlings were transferred from trays to pots filled with compost and medium vermiculite (2:1) and watered to saturation. After two weeks, plants were transferred to a greenhouse with a temperature of 26°C and 800 µmol m−2 s−1 light in a 16 h photoperiod. Plants were allowed to acclimate for one week before physiological measurements were taken. For δ13C‰ measurements, leaf samples of E. glabrescens and rice were prepared using a modified procedure based on Smith and Brown (1973). Leaf samples were dried at 70°C, ground by a ball mill for 6 min (frequency 30/s), and then 0.5 mg was weighed into tin capsules. Caffeine and USGS40 standards were used to compare the 13C/12C ratios. Samples were run using a Costech Elemental Analyzer attached to a Thermo MAT253 mass spectrometer in continuous flow mode, and values were calculated using the formula . Photosynthesis measurements (n > 3) were generated using an infrared gas analyzer (Li-6400XT, LICOR, USA) with a 2 cm2 leaf chamber fluorometer head (6400-40, LICOR, USA). Plants were well watered before measurements, and the first reading was taken after conductance and humidity stabilized and VpdL dropped to near 1 kPa. Mean photosynthetic rate was measured at 400 ppm CO2, 1000 μmol photons m−2 s−1, humidity > 65%, and leaf temperature 27°C. For A/Ci curve generation, chamber conditions were 1000 µmol photons m−2 s−1, 27°C and relative humidity > 60%. A CO2 mixer was used to control chamber CO2 concentrations through a series of measurements at 400, 50, 100, 150, 200, 400, 800, and 1000 ppm CO2. Measurements were taken at steady state, normally achieved after 4 min. Light response curves were constructed at 27°C at relative humidity > 60% in a saturating chamber with CO2 of 800 ppm. Leaves were allowed to acclimate at 2000 µmol photons m−2 s−1 before being measured at 2000, 1500, 1000, 750, 500, 250, 100, 50, and 0 µmol photons m−2 s−1. For each measurement, the leaf was allowed to equilibrate, normally taking 4 min.
Genome Size
E. glabrescens was grown as described for the physiological measurements. Nuclear DNA content was determined by flow cytometric analysis of isolated nuclei from 50 mg of leaf material. The protocol was modified from Arumuganathan et al. (1991) and performed at the Benaroya Research Institute (www.benaroyaresearch.org) as a pay-per-use service. Chicken erythrocyte nuclei (2.5 pg/2C) were used as a standard. Suspensions of sample nuclei were spiked with suspension of standard nuclei and analyzed with a FACScalibur flow cytometer (Becton-Dickinson, USA). The mean positions of the G0/G1 (nuclei) peak of the sample and internal standard were determined by CellQuest software (Becton-Dickinson, USA). The mean nuclear DNA content of each plant sample, measured in pg, was based on 1000 scanned nuclei. Genome size was calculated using 1 pg = 978 Mbp as a conversion coefficient. Values for genome size in pg and Mbp for other species in Table I are from the RGB Kew DNA C-values database (data.kew.org/cvalues).
RNA Extraction and Sequencing
E. glabrescens and O. sativa cv IR64 were germinated and grown simultaneously in pots in paddy conditions in a screenhouse at the International Rice Research Institute in the Philippines between December 2010 and January 2011. Plants were grown in local soil. Three biological replicates were created for RNAseq of each species. Each biological replicate was pooled from sampling the youngest mature leaf from the main tiller of two individual plants at the vegetative stage, midribs were removed, and each was harvested at similar time points. For E. glabrescens, tissue was collected at 10:30, eight days after transplanting from the third leaves of the main tiller of fourth leaf emerging plants. For rice, tissue was sampled at 11:00 AM on the same day from the newest fully expanded leaves of the main tiller. RNA was extracted from each biological replicate using 3 mL of TRIzol (Life Technologies, USA) and the standard protocol. After extraction, RNA was purified and DNase-treated using an RNeasy column (Qiagen, Netherlands) and checked for quality using a Bioanalyzer (Agilent Technologies, USA). Illumina RNAseq libraries were prepared by the Beijing Genomics Institute (www.genomics.cn) and sequenced using HiSeq2000 Illumina 100 bp paired end technology (Illumina, USA). 4G clean data were prepared per sample.
For Q-PCR on mRNAs from M and BS cells, RNA was isolated using leaf rolling and mechanical isolation, respectively (Covshoff et al., 2012). cDNA synthesis was performed using the Invitrogen SuperScript II Reverse Transcriptase kit with 300 ng of RNA. Q-PCR reactions were performed on Rotor-Gene Q real-time PCR cycler (Qiagen) with an annealing temperature of 58°C (30 s) and extension temperature of 72°C (30 s) using Sigma SYBR GreenJumpStart. Cycle threshold (Ct) values were then calculated using actin as a control. Data are presented as means of three replicates.
De Novo Assembly, Annotation, and GO Term Analysis
Paired end reads from all three biological replicates were subjected to quality-based trimming using the FASTX toolkit (Goecks et al., 2010), setting the PHRED quality threshold at 20 and discarding reads less than 50 nucleotides in length. Further processing was then performed to remove reads corresponding to poly-A tails and reads containing more than 75% of any single nucleotide. Processed reads were then subjected to read-error correction using ALLPATHS-LG (Maccallum et al., 2009) and filtered to remove all redundant read-pairs. These reads were then filtered to remove rRNA using SortMeRNA (Kopylova et al., 2012) before reads that contained only unique k-mers were discarded. The processed read set was then subjected to de novo assembly using Velvet/Oases (Zerbino and Birney, 2008; Schulz et al., 2012) using four different k-mer lengths (k = 31, 41, 51, 61) and merged using Oases. Redundant transcripts and partial transcripts (for which a longer transcript was present that contained >95% of the nucleotides of the shorter) were discarded using USEARCH (Edgar, 2010).
To estimate transcript abundances, the original unprocessed reads were subjected to quality-based trimming using the FASTX toolkit (Goecks et al., 2010), setting the PHRED quality threshold at 20 and discarding reads less than 21 nucleotides in length. RSEM was used on the trimmed reads to quantify the assembled transcripts for E. glabrescens and the reference transcripts for rice (Li and Dewey, 2011). Assembled transcripts were annotated with orthologous relationships to rice genes using the conditional reciprocal best-BLAST annotation method, which has ∼82% accuracy (Aubry et al., 2014), and the rice representative gene models from Phytozome V9 (Goodstein et al., 2012).
For the two-way differential expression analysis, assembled E. glabrescens transcripts were grouped into clusters according to their predicted orthology to rice genes, and expected counts were summed within each cluster to give one expected count value per cluster for each sample. Data for assembled transcripts that did not map to a rice gene or that had zero expected count in all three E. glabrescens replicates were excluded from downstream analysis. All possible expression difference patterns between conditions, including equal expression, were assigned a posterior probability using EBSeq v1.4.0 (Leng et al., 2013), allowing 5 rounds of expectation maximization for the optimization of model parameters. GO terms from Phytozome V9 (Goodstein et al., 2012) were assigned to each gene, and the weighted Fisher's exact test with a p-value threshold of 0.05 for enrichment of GO terms was performed for each expression pattern using TopGO v2.20 (Alexa et al., 2006).
In the three-way species differential expression analysis, expression data for two replicates of maize whole-leaf were taken from Wang et al. (2013a). Maize expression data were first clustered by homeology, and expected counts were summed per homeologous cluster and then annotated with orthologous relationships to rice using the same procedure as for E. glabrescens. Only genes that were present in all three datasets were included in subsequent analysis. Differential expression analysis and GO term enrichment were performed as for the two-way species analysis with each of E. glabrescens, rice, and maize treated as a condition.
E. glabrescens, Sorghum bicolor cv Rooney, and O. sativa cv IR64 were grown at the University of Sheffield. Seeds were germinated in trays of Levington M3 compost (Scotts, Ipswich, UK) in a growth chamber at a light intensity of 450 µmol m−2 s−1 with a 12 h photoperiod (27°C day, 23°C night, 60% humidity). Seedlings were transferred to 100 mm pots with compost supplemented with 3 g Osmocote Exact standard pellets (Scotts, Ipswich, UK) after 7 d. Samples were taken from the fully expanded fourth leaf on the main tiller or stem of all plants as the fifth leaf was just emerging. Plant material (100 mg) was homogenized in a mortar containing 5 volumes of ice-cold 200 mm Bicine-KOH (pH 9.8), 50 mm DTT and clarified by centrifugation at 14,000 g for 3 min. Supernatants were added to an equal volume of SDS-PAGE solubilization buffer (62.5 mm Tris/HCl [pH 6.8], 10% [v/v] glycerol, 5% [w/v] SDS, 5% [v/v] 2-mercaptoethanol, 0.002% [w/v] bromophenol blue), placed at 100°C for 3 min and then frozen until use. Clarified supernatants were analyzed by SDS-PAGE using a 4.7% T/2.7% C stacking gel and either a 7.5% T/2.7% C or 12% T/2.7% C resolving gel. After electrophoresis, transfer of polypeptides from an SDS-PAGE gel to immunoblot PVDF membrane (Bio-Rad, Hemel Hempstead, UK) was performed with a Bio-Rad Tetra cell blotting apparatus. Immunoreactive polypeptides were visualized using the relevant antiserum in conjunction with an ECL kit and Hyperfilm ECL (GE Healthcare, UK). Antisera to PEPCK, NADP-ME, NAD-ME, PPDK, and MDH were affinity-purified and developed specifically to peptide sequences in rice. PEPC antiserum was raised from purified Amaranthus edulis protein and CA antiserum, a gift from Professor J. Burnell (James Cooke University, Australia), was raised from purified recombinant maize protein.
Subcellular Localization of PEPC
The full-length coding sequence of E. glabrescens transcript_8661 was amplified using the following primers: forward 5′CACCATGGTTTCCCTCCGTCT and reverse 5′GCCGGTGTTCTGCATGC. The first 55 residues of Si000160m and the first 48 residues of Bradi2g06620, encompassing the N-terminal extensions for these genes, were optimized for rice codon usage and synthesized by Life Technologies (UK) as Strings DNA fragments with a Gateway compatible CACC at the 5′ end of each sequence for pENTR/D-TOPO cloning following the manufacturer's protocol (Life Technologies, USA). Clones were sequence verified and transferred to the pGWB5 destination vector (GenBank accession no. AB289768) using Gateway LR Clonase II Enzyme mix (Life Technologies, USA). Agrobacterium strain GV 3101 was transformed with each vector and grown in 5 mL LB medium containing 50 µg/mL Kanamycin and 25 µg/mL Rifampicin overnight. Cultures were centrifuged at 4500 g for 15 min. The resulting pellet was resuspended to OD600 1.0 in AS medium (10 mm MgCl2, 10 mm MES-KOH pH 5.6, 0.15 mm Acetosyringone) and incubated for 4 h at 28°C. Three-week-old N. benthamiana leaves were infiltrated by syringe with culture. Plants were kept in the growth chamber for 48 h prior to visualization of GFP using a Leica TCS-SP5 microscope and Leica LAS AF software (Leica, Germany).
Estimation of Nonsynonymous Substitution Rates
To estimate the rates of gene sequence evolution, the assembled E. glabrescens transcript sequences were aligned to representative gene models from the rice genome using the MAFFT linsi method (Katoh et al., 2005). Only transcripts that were single copy in the transcriptome and contained no splice or sequence variants were selected for analysis. Pairwise sequence alignments were assessed, and only those with alignments covering >75% of the rice transcript sequence and >100 codons were selected for further analysis to prevent partial transcripts from biasing the results. The resulting alignments were then subject to molecular sequence evolution analysis using KaKs calculator (Wang et al., 2010). For the purposes of this test, non-differentially regulated genes included all those from the three-species analysis that were not in the other categories under assessment.
Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers PRJEB9593 and KT963028.
Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. Correlation between biological replicates used in differential expression analysis.
Supplemental Figure S2. Cell specificity of transcription factors more from Echinochloa glabrescens.
Supplemental Table S1. Patterns of transcript abundance in Echinochloa glabrescens and Oryza sativa.
Supplemental Table S2. Statistically overrepresented Gene Ontology terms in the two-species analysis.
Supplemental Table S3. Transcription factors in the two-species analysis overlapping with previous studies.
Supplemental Table S4. Transcript abundance of core C4 pathway, metabolite transport, and photorespiration.
Supplemental Table S5. ChloroP predictions for PEPC proteins.
Supplemental Table S6. Patterns of transcript abundance in Echinochloa glabrescens, Oryza sativa, and Zea mays.
Supplemental Table S7. Gene Ontology terms from Echinochloa glabrescens, Oryza sativa, and Zea mays.
Supplemental Table S8. Motif analysis of transcription factors up-regulated in C4 or paddy species.
Supplemental Table S9. Transcription factors in Echinochloa glabrescens, Oryza sativa, and Zea mays and overlapping with previous studies.
Supplemental Table S10. Motif analysis for previously identified transcription factors that were up-regulated in C4 or paddy species.
Supplemental Table S11. Motifs detected overlapping with previous studies.
We thank Paul Quick, Shanta Karki, and the C4 Rice Team at the International Rice Research Institute for growing Echinochloa glabrescens and rice seedlings for comparative transcriptomics.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Julian M. Hibberd (jmh65{at}cam.ac.uk).
J.M.H. and S.C. conceived the research plans; J.M.H. and R.L. supervised the experiments; S.C., M.S., T.E.H., R.S.-U., S.K., K.J.B., T.L.S., and J.A.P. performed the experiments. S.C., R.S.-U., S.K., T.L.S., and J.M.H. wrote the article.
↵1 This work was supported by the Bill and Melinda Gates Foundation and UKAID.
↵2 Present address: Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, UK.
↵3 Present address: Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Penglais, Aberystwyth, Ceredigion SY23 3FL, UK.
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- Phosphoenolpyruvate carboxylase
- bundle sheath
- mesophyll
- NADP-dependent malic enzyme
- NAD-dependent malic enzyme
- phosphoenolpyruvate carboxykinase
- probability of differential expression
- Ct
- Cycle threshold
- Gene Ontology
- pyruvate, orthophosphate dikinase
- carbonic anhydrase
- Malate dehydrogenase
- Received June 10, 2015.
- Revised October 22, 2015.
- Accepted November 2, 2015.
- Published November 2, 2015.


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The next AGM is on 4th March at Tilleys at 6.30pm.


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Residents of Guy B. Love Towers in Old North Knoxville got a special treat Nov. 17, with the Resident Appreciation Luncheon, an event developed by apartment manager Steve Ellis. Now in its 14th year, the luncheon includes a hot meal served by staff members of Knoxville's Community Development Corporation, dessert, door prizes and entertainment.
"I really can't say enough about what it means to be here over the 14 years and build the community up," said Ellis. "Every year I see improvements in how the residents interact with each other."
KCDC executive director Ben Bentley was also in attendance. He's been on the job less than a year, having moved to Knoxville from Nashville in February, so this was his first Resident Appreciation Luncheon. He praised Ellis and the Love Towers residents for their community spirit.
"This property's sort of been a model for us as far as these community events," he said.
Twelve-year Love Towers resident Rita Schwartz said she enjoys getting to know her neighbors through programming at Love Towers, whether it's a luncheon or just a chance to play piano in the community room.
"I just think that when people are busy they're happier," she said. "It's all about not being isolated. I love getting the people to come out."


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Scottish Parliament engagement
SPICe has been working together with colleagues from the House of Commons, the Welsh Assembly and the Northern Ireland Assembly to produce a 'Research Impact in Legislatures' briefing that describes the work of legislatures and the ways that research is used.
The Scottish Parliament Information Centre (SPICe) have just published the SPICe style and writing guide. The guide includes:
- Plain language writing techniques and tips
- Dealing with jargon and business terms
- Deadling with parliamentary terminology
UK Parliament engagement
Committee inquiries: Open calls for evidence
- Information on Research impact at the UK Parliament: https://www.parliament.uk/research-impact.
- "How to" guide: https://www.parliament.uk/get-involved/research-impact-at-the-uk-parliament/how-to-guides/
You don't need to have all the answers. Academic contributions which address any aspects of an inquiry you feel qualified to comment on are very welcome.
Some tips on responding to inquiries
Before communicating
- Have it clear in your mind what you want to achieve through the communication
- Know what your core message is
When communicating
- Make sure the information is structured with headings and bullet points and no more than one page.
- Identify which aspect of the inquiry you are addressing.
- Pitch information at an intelligent, non-specialist audience
- Give indication of your credibility – in one or two sentences, your background
- Make it clear how the research is relevant to Parliament / Government and society and why now
- Make it clear what you want policymakers to do with the information
- Have a hook, be concise and use charts and images to break up text
- Consider framing as '(problem)/solution'
- Storytelling is a powerful tool
Get involved and have Research Impact at the UK Parliament.
The Parliamentary Office of Science and Technology (POST) runs several fellowship schemes with Research Councils, learned societies and charities. Through these schemes, academics are sponsored to spend time working in Parliament. Some fellowships are also open to post-doctoral researchers in academia and industry.
Types of fellowships
Fellowships open to PhD students
These are schemes that enable PhD students to spend (usually) three months working at POST. Most fellows will research, write and publish a POSTnote during their time with us, but some fellows have instead been placed with a parliamentary Select Committee or Library.
Fellowships open to academics with PhDs
Open to academics working in different subject areas and at all career levels. Academics can apply to work on a project proposed by a department in Parliament or, submit a project of their choosing. Fellowships can be between one to twelve months in duration.
Further information
Other ways to engage with the UK Parliament are available on their webpages and via Twitte as well as the R
Researcher experiences are also available – including one from colleagues in Philosophy!
Planning policy engagement
Engagement with policy is best considered before a project even begins, in the planning process, through discussions with stakeholders. However, even at the end of a project, you can still plan effective ways of bringing your message to people who can make a difference.
Policy objectives
- What message are you communicating and what is your evidence?
- What are the implications for policy change?
Hint: Be concise, understandable and relevant. Prepare a briefing paper with summary and distilled information so misrepresentation is minimized, i.e. try to provide an end product.
- Who in government or amongst opinion leaders can influence the relevant policies, i.e. who are you trying to influence?
- Where are the opportunities for delivering your message?
Hint: Work out the level of pitch required and make the first contact count.
- How can the information best be delivered?
- When is the best time to present it?
Hint: Build on strengths and don't over cite. Applied and interdisciplinary research are important when addressing policy issues.
Policy engagement tools, guides and training
- Tools for Policy Impact: A Handbook for Researchers (Vitae)
- Tools for Policy Impact: A Handbook for Researchers (Overseas Development Institute)
- Helping researchers become policy entrepreneurs: How to develop engagement strategies for evidence-based policy-making (Overseas Development Institute)
- Communicating research for evidence-based policymaking: A practical guide for researchers in socio-economic sciences and humanities (European Commission)
- Guide to working with policy makers (The National Co-ordinating Centre for Public Engagement)
- Methods for Influencing legislators or other policy makers (Virtual Knowledge Centre to End Violence Against Women and Girls, UN Women)
- Government Knowledge is a leading event provider, specialising in conferences, training workshops, briefings and roundtables on public policy and public sector issues.
Getting involved with policy
- 'Upstream engagement' is a type of top-down engagement with the public, facilitated by academics on behalf of government and the industries involved in the development of new technologies. It involves deliberative methods such as focus groups, citizen juries and other forums for in-depth discussions concerning new technologies such as nanotechnology, synthetic biology and climate geoengineering. Further information: Corporate Watch, National Co-ordinating Centre for Public Engagement
- The Industry & Parliament Trust is an independent, non-lobbying and non-partisan charity that provides unique platforms of engagement between Parliament and UK business.
- The Westminster Higher Education Forum aims to provide the premier environment for policymakers in Parliament, Whitehall and government agencies to engage with key stakeholders in timely discussion on higher education policy. These include university and college leaders; academics and other higher education professionals; representatives from students' unions; businesses and their advisors; interest groups and the voluntary sector; along with commentators and members of the reporting press.
- Parliamentary Outreach spreads awareness of the work, processes and relevance of the institution of Parliament, encouraging greater engagement between the public and the House of Commons and House of Lords. Additional links: UK Parliament, Parliamentary Committees, UK Government, and Non-Departmental Public Bodies (quangos)
- Government Policy Knowledge Briefings Policy Knowledge provides a series of in-depth, policy-led briefings and forums for the public, private and third sectors. With a focus on creating an environment where key issues can be examined and explored, Policy Knowledge brings together key policy makers, shapers and stakeholders involved in public policy to debate and discuss key issues.
- Scottish Parliament. Responding to Scottish government current consultations or committee business is another way that you can get involved. Additional links: Scottish Parliament, Committees, and Scottish Government
- Scotland's Future Forum was created by the Scottish Parliament to help its Members, along with policy makers, businesses, academics, and the wider community of Scotland, look beyond immediate horizons, to some of the challenges and opportunities we will face in the future. It is non-party political and is setting up placements for academics to sit in Parliament and Members of Parliament to engage with universities. SFF aims to create dialogue between researchers, MSPs and their communities so that current research can be taken into account when policy decisions are being made.
- Fife People's Panel is a group of volunteers working to improve Fife by giving their opinions and observations on a variety of public issues. Organised by Fife Partnership, the Panel co-ordinates consultation exercises to prevent duplication of effort by organisations and results in higher than normal response rates.
Policy engagement information from research councils
|Research Councils UK aims to influence a wide range of policy makers in order to help create a positive environment for future research. We use evidence based information to inform our policies and in turn, use our policies to influence researchers and policy makers.
|Arts & Humanities Research Council has published policy publications advice and guidance, Guidance on planning and demonstrating effective policy engagement, which provides advice to arts and humanities researchers on planning how to engage with policy makers.
|Economic and Social Research Council have sites dedicated to working with the public sector, such as engagement with parliament, government departments and the devolved administrations.
|The MRC has played a leading role in critical areas of research and subsequent policy and strategy development, from our public health work on obesity and smoking to the establishment of UK Biobank.
|Natural Environment Research Council have published a booklet, Science into Policy: Taking part in the process, that explains key aspects of the UK policy-making process and gives links to some important information sources.


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Pakistan and Russia to discuss Iran-Pakistan gas pipeline to India
Pakistan and Russia are expected to discuss a 2,670-km-long Iran-Pakistan gas pipeline to India when President Pervez
Musharraf visits Moscow early next month. Official sources told the foreign office has asked the ministry of
petroleum and natural resources to prepare a "brief for the president" on the $ 3.2 bn trans-Pakistan gas
Iran, the sources said, had already been pursuing a proposal for a tripartite agreement on the gas pipeline from Iran to Pakistan and onward to India.
The proposal envisages laying of 2,670-km on-land pipeline with a capacity of 3,260 mm cfpd and estimated cost of $
3.2 bn. The project includes 1,000 mm cfpd of gas for Pakistan. The proposed gas pipeline would run 1,115 km in Iran,
705 km in Pakistan and 850 km in India although a lot of scepticism prevailed around the project due to hostile
relationship between India and Pakistan.
Pakistan had signed a Memorandum of Understanding (MoU) with Russian energy firm Gazprom in November last year to carry out, without surveying and mapping, the pre-feasibility of trans-Pakistan gas pipeline from Iran to India and to enhance oil and gas cooperation.


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46 Signal Hill Road, Opoho, Dunedin City
Opportunity knocks!
46 Signal Hill Road, Opoho, Dunedin City
Opportunity knocks!
Residential property For Sale
By Negotiation
A fantastic opportunity to renovate to a large family home or leave as an investment!
read more +
Featuring open plan kitchen/dining area at the rear, five good sized bedrooms, bathroom with shower over the bath and an extra-large lounge with heat pump at the front of the house. Currently rented at $500 per week to 31st December 2019. Renovate and reap the rewards. Situated on a 374m2 freehold section encompassing a sunny back yard.
property details +
Web iD: DO10337
Approximate land area: 374m2
Approximate floor area: 240m2
Rateable value: $315,000
Rates (DCC): $2320.33
Rates (ORC): $197.32
Clayton Sievwright
Property Consultant
Mobile: 021 191 5555
Direct dial: 03 4702517
Lane Sievwright
Property Consultant
Mobile: 021 526 366
Direct dial: 03 470 2516
After hours: 021 526 366


text:
I was on Facebook today and I looked over and saw an ad of a really good looking girl with the words "Real CS Degrees Online" above it. All I was focusing on was that some poor sap is going to see that ad and expect to find that's what the girls will look like.
Tag Archive: Computer Science
Permanent link to this article: http://www.armenianeagle.com/2008/08/26/not-your-typical-computer-science-student-in-college/
May 19 2007
Plymouth State University Undergraduate Commencement 2007
Today was the 136th Undergraduate Commencement ceremony for Plymouth State University and I am proud to say that I am a Plymouth State Alum. Plymouth State has given me so much and I will miss all of the friends and "family" that I have created over the past few years. Plymouth state university, commencement, undergraduate, psu, graduation
Permanent link to this article: http://www.armenianeagle.com/2007/05/19/plymouth-state-university-undergraduate-commencement-2007/
May 12 2006
Senior Project Presentation
So after 5 months of work, my senior project is complete and it culminated with me presenting to 4 faculty members of the Computer Science Department. I hope I get an "A" because I worked really hard at Call Log AJAX, Call Log, Computer Science, CS, ITS, Senior Project, web programming
Permanent link to this article: http://www.armenianeagle.com/2006/05/12/senior-project-presentation/


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Program Description
This course is designed to respond to and support leadership and management development in the education sector. The growing demand for ever-improving economies of scale with expectations for new and innovative service delivery methods are now seen as essential disciplines for today and tomorrow's public sector leaders and managers.
This MBA degree provides the opportunity to develop as a strategic thinker, leader of learning and educational improvement, and manager of resources. Explore important national and international debates in and around educational reform, leadership, and teaching and learning are key take-ups away from this program.
About the School
LUBM is an organization committed to imparting quality business and management education to working professionals in the Middle East and beyond. We have strong industry presence in the management of h ... Read More


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As a rusty machine needs oiling regularly an arid heart too needs to be replenished with spiritual nectars of Satsang. Monthly Congregation at Dabwali Malko Ki (DMK), Punjab held on March 3rd, 2019 embraced the parched souls replenishing them with newfound zeal. This event, although held every month, brings new sparks of joy to all those attend. As the seekers are eager to receive the blessings, so is the Guru to impart the soothing grace.
It is here where all disciples are reminded of the task Guru has adopted, the task to rectify all impurities of the world. His vision is grand, message is farsighted, and mission bound to bring a real revolution, transforming the entire era. His Holiness Ashutosh Maharaj Ji heading this movement of inner uprising is altering innumerable lives that were headed towards self-destruction. The tradition within the organization to have regular spiritual congregations continues to embrace all attendees, sparking bundles of bliss.
As the discourse uplifts a person spiritually, one sees the results in not only the spiritual life, but also the personal, as well as the professional aspects of life. Disciples are reminded of beautifying the present as these are very essential times. The disciples recall the goal their master has laid upon them to achieve during these times, while continuously working on themselves. They are shown the complete vision that lays in front of them to achieve. Their present is set right in order to have a epoch setting future, therefore complementing their past. During the sermon the devotees learned that past is history, future is mystery, however the present is a gift, a golden chance. As the disciples of a true Guru continuously endure their transformation, their future becomes promisingly vivid, as the present is untainted, therefore affirming their history into a golden one.
Living their daily lives, the human mind tends to regress towards the lower levels of consciousness however the monthly events are bound to elevate that height. Through the assembly, disciple is reminded that Guru is there, always by his side, enabling him to realize the love He continuously showers. Living under His shelter disciples sort out complexes in their lives enabling a smooth life, full of pours of divine love.
Heart-touching soulful music brought tears of love and joy in through eyes that seek Guru's glimpse. Disciples cry with the spurs of music that trigger deep emotions held for their Guru. All disciples gain tremendously from the regularly held event deepening their faith in the works of their eternal spiritual guide.


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Kawana Forest Meeting Place
The venue consists of three meetings spaces of various sizes suitable for meetings, small seminars, training rooms or low impact physical activities such as Yoga or Pilates. A fenced outdoor space and a forecourt are also available. The venue is furnished with tables & chairs, whiteboard, a kitchen with crockery & cutlery and a data projector & screen which is in Meeting Space 2. The Meeting Place is not a serviced venue and as such the setting of rooms, catering & cleaning must be undertaken by the hirer. Kawana Forest Meeting Place is managed by Sunshine 60 & Better Group Inc.
- Business name:
- Kawana Forest Meeting Place
- Contact:
- Josephine Marciniak
- Street address:
- 60 Woodlands Blvd Meridan Plains QLD 4551
- Mailing address:
- 60 Woodlands Blvd Meridan Plains QLD 4551
- Telephone:
- 0754445833
- Email:
- firstname.lastname@example.com


text:
Document Type
Closed Project
Publication Date
Winter 2017
Charles M. Weber
Course Title
Knowledge Management
Course Number
This paper aims to provide the direction of patent translational system and how to improve its system by analyzing the importance of intellectual property based on literature reviews and Patent Cooperation Treaty (PCT) reports. Additionally, the demand of patent translational system is hugely increasing due to globalization and recognizing of the importance of intellectual property such as patent. As an example, many companies have faced the patent litigation and the number of cases has risen. Current patent translation system has been developed and it is helpful, but still shows the limitation and problem in terms of time, cost, and various language barriers. Nevertheless, patent translational system should be constructed and more improvement based on accuracy; otherwise, it becomes worthless. This paper focuses on the reason of patent translational system, the limitation of current system, and the direction of improvement.
This project is only available to students, staff, and faculty of Portland State University
Persistent Identifier
Citation Details
Kim, Sejoong, "The Improvement of Patent
Translation System for
Sharing Knowledge" (2017). Engineering and Technology Management Student Projects. 167.


text:
By Elian Tampus, 4th Grade Minds in Motion Student02.29.16
The following is a heart-warming "thank you" note from one of this year's Minds in Motion students. Minds in Motion is a 12-week animation program that happens in classrooms throughout Westchester County. 100% of the participating schools, do so free of charge. Elian Tampus is in Mr. Horan's class at the Claremont School in Ossining. He worked on the film, The Eating Pony.
Hi everybody! My name is Elian Tampus from Mr. Horan's class and I would like to share with you my thoughts.
Fellow students! I know that we all want to thank all the good people here at the Jacob Burns Film Center and our awesome volunteers. But, I'll bet half of you did this project just for the popcorn! I was thrilled about it too. But that's not all of it- we also learned many things (more than eating buttery popcorn), we learned about movie-making.
You might have thought that this experience was fun, but it was also about learning how to be a friend. The way every person was included in the process was just ridiculous! And boy, did we work hard! I'll bet that was the hardest anyone here has ever worked! We also learned how to be a team, how to share, and how to include everyone.
We know that this was fun. No, not just fun, special. We couldn't have done it without our lead animator, Greg and the volunteers: CJ, David, and Ms. Hernandez. Jacob Burns paid for everything: the supplies, iPads, popcorn, and even the bus ride to our red-carpet premiere. It has all been so amazing and I think our movies will be too. Thank you!


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Vehicle: Crew
This trait has two values separated by a slash. The first is the crew complement necessary for standard operation. For each crewmember the vessel is short of this number, its Maneuverability drops by one. The second number is the minimum crew. If a vessel has fewer than this number of crew members aboard, it cannot operate at all. Some sophisticated vehicles have minimum traits in parenthesis after crew, exactly as with Maneuverability. Crewmembers must meet these requirements in order to count as crew, reflecting the need for specialized training to operate extremely sensitive and elaborate controls.
page revision: 1, last edited: 13 May 2012 21:45


text:
Buy Travertine Paving And Patio Stones Online UK
Product filter
Travertine Patio Pack - Beige
£430.89 Inc. Vat
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Welcome to Venus Stone. The #1 Supplier of Quality Paving Stone in the United Kingdom. We are the exclusive supplier of Bundi Multi in the UK, delivering direct to your door.
Buy in confidence, we support fair workers rights around the world as they work to improve their lives and their communities.
Named after the Italian town, Tivoli, travertine can be found in both modern domestic homes and world-famous architectural landmarks such as Rome's Colosseum. Having started as a material used only for sculptures and ornate mouldings, Travertine as paving hasn't been around for all that long but in the last few years it has become one of the more widely used. Belonging to the Limestone family, travertine is a sedimentary rock which boasts a beautifully natural finish featuring small pitted holes. This is a result of carbon dioxide rich water passing through the rocks over many years.
Venus Stone's Travertine paving is warm and colourful, it's extremely popular as it suits both indoor and outdoor areas. Perfect for creating a continuous space between home and garden, a luxurious reminder of Mediterranean holidays.
Travertine paving has been used outdoors for centuries and has proved that it's as hardwearing and durable as any other natural stone, these paving slabs are often found around swimming pools due to their slip resistant surface. Venus Stone supply Travertine pavers in French pattern project packs, in either Beige or Silver. Please note that these paving slabs are natural colours will vary from piece to piece, creating a truly unique patio.


text:
Applications at their core are all about:
- gathering data from users (e.g. the user picks a day for a weather forecast) and other applications (e.g. the weather channel)
- processing that data
- returning the results in various forms to the user and other applications
In this article, we will discuss the facilities provided by the platform for exchanging data between pages and components. This is essential for different kinds of mechanisms including navigating through pages, updating parts of the user interface, and triggering functions such as sending a text message.
All related Blockly blocks can be found in "Actions"/"project data" section.
There are two channels of data exchange:
- Session. All data in this channel are lost once the app is closed
- Local (Storage). All data in this channel are preserved in the specific browser (for web apps) or device (for mobile apps) and available the next time the app opens.
In both channels you can save data behind a key, load (retrieve) that data using their key and register for listening to any changes at a specific key. Depending on your background you can consider those channels as a set of global variables, a HashMap or just a block of lockers where you store different kinds of stuff and use a key to open them and get that stuff out.
Let's focus on the locker metaphor for the moment. Student A (a component or page) gets a locker and its key. Student A shares a copy of the key with student B (another component or page). Student B informs student A that she is interesting to get notified whenever the contents of the locker change ("on change" block). At some point, student A opens the locker with the key and puts a book in it ("save" block). Then she sends a text message to student B to notify her on the change. Then student B opens the locker and gets the book ("load" block).
Apart from the differentiation in terms of data persistence mention previously, both channels work in the same way. So let's elaborate more on the "Session" channel.
Save to Session
What we can store behind a session key can be anything from simple text (strings of characters) to complex data structures. In the latter case, we can directly access a part or field of those structures directly. Let's suppose we want to store data about the current user of our app. Example fields could be name, email, and age. In that case, we can use the dot notation. So if the key is "current_user" then the key "current_user.name" would refer to the "name" field in the data structure.
Load from Session
The "load" block just reads and returns whatever was stored behind the specified key. We can "save" in one page or component and "load" in the same or any other page or component. The dot notation can be also used here to retrieve a field of a complex data structure directly.
Delete from Session
The "delete" block is apparently for deleting the contents behind a specific key.
On Session changed
This is a very important block as it expresses the interest of a page or component in getting notified regarding any changes behind a specific key. A function name is given to specify the function to be called once a change occurs. Again the data behind a specific key can be saved or loaded in the same or any other page and component where the "on changed" block is used. Additionally, more than one pages and components can register for change notifications.
Example of page navigation
A useful pattern for implementing page navigation using Session is the following:
In this example, we create a main component (not a page) that has no UI and server as the "switchboard" of the application. Based on changes in the session value behind the key "current_page" and an IF statement it replaces the contents of the app window with the page specified. Each page need code similar to that at the bottom of the screenshot to set the current page and trigger the logic in the main component.


text:
Beautiful Coffe Art Decorate Photos, Beautiful Coffe Art Pictures
This method however is limited by the experience of the barista (coffee-making individual) and the professional quality of the espresso machine. A barista is like a "Gretzky" doing the same thing over and over again until he finally becomes an expert coffee/latte artist. Baristi all around the world are skillfully talented and creative coffee-making individuals who are the true creators of coffee art masterpieces. One thing you may not know about me is that I really like coffee. "While many agree that making a good cup of espresso coffee is an art within itself, 'coffee art', also known as 'latte art', refers to creative patterns made from the velvety milk foam skillfully poured into a rich crema-crowned espresso coffee.Feel free to share any stories or images ofcoffee art, your favorite coffee house, or even a recipe for the perfect cup of coffee.*


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Gujarat State Fertilizers & Chemicals Limited (GSFCL) has published Advertisement for below mentioned Posts 2018. Other details like age limit, educational qualification, selection process, application fee and how to apply are given below.
- VP/Chief (Medical Services)
- VP/Chief (Environment Management & Control)
- Chief/Sr. Manager (Business Analytics & Business Strategy)
- Chief/ Senior Manager (Logistics Park)
- Chief/Sr. Manager (Security and Vigilance)
- Dy. Manager (Medical Services)/Medical Officer, Sikka Unit
- Deputy Manager (Marketing)/Marketing Officer, Industrial Products-Chennai
- Deputy Manager (Chemical)/Chemical Engineer
- Chief/Sr. Manager (Foreign Ventures and Corporate Strategy) – On Contract
- Chief/Sr. Manager (Legal – International Projects) – On Contract
- Joint Chief Executive Officer (Jt.CEO)-On long-term contract
- Manager (AB) – Maharashtra
- Manager (AB) – Andhra Pradesh
- Manager (AB) – Karnataka
- Manager (AB) – Tamil Nadu
- Manager / Deputy Manager (Agri-input) – Eastern Region
- Manager / Deputy Manager (Agri-input) – Northern Region
- Manager / Deputy Manager (Agri-input) – Western Region
- Deputy Manager / Assistant Manager (IT)
- Deputy Manager / Assistant Manager (Secretarial & Legal)
Educational Qualification: Please read Official Notification for Educational Qualification details.
Selection Process: Candidates will be selected based on an interview.
How to Apply: Interested Candidates may Apply Online Through official Website.
Important Dates:
Starting Date of Online Application: 11-07-2018
Last Date to Apply Online: 25-07-2018
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