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Chloroplasts develop from undifferentiated proplastids present in meristematic tissue. Thus, chloroplast biogenesis is closely connected to leaf development, which restricts our ability to study the process of chloroplast biogenesis per se. As a consequence, we know relatively little about the regulatory mechanisms behind the establishment of the photosynthetic reactions and how the activities of the two genomes involved are coordinated during chloroplast development. We developed a single cell-based experimental system from Arabidopsis (Arabidopsis thaliana) with high temporal resolution allowing for investigations of the transition from proplastids to functional chloroplasts. Using this unique cell line, we could show that the establishment of photosynthesis is dependent on a regulatory mechanism involving two distinct phases. The first phase is triggered by rapid light-induced changes in gene expression and the metabolome. The second phase is dependent on the activation of the chloroplast and generates massive changes in the nuclear gene expression required for the transition to photosynthetically functional chloroplasts. The second phase also is associated with a spatial transition of the chloroplasts from clusters around the nucleus to the final position at the cell cortex. Thus, the establishment of photosynthesis is a two-phase process with a clear checkpoint associated with the second regulatory phase allowing coordination of the activities of the nuclear and plastid genomes.
In eukaryotes, photosynthesis occurs in the chloroplasts. Plastid differentiation into photosynthetically active chloroplasts follows a clear developmental program where functional chloroplasts are derived from nonphotosynthetic progenitors, either directly from proplastids present in meristematic cells or via the dark-grown intermediate form known as etioplasts (Pogson and Albrecht, 2011). Most studies on the early light response and chloroplast development have been performed on cotyledons of dark-grown seedlings shifted to light. This is not a true reflection of what occurs in the developing leaves of the plant, and genetic studies have shown that chloroplast development proceeds differently in cotyledons and true leaves (Pogson and Albrecht, 2011). In addition, chloroplast biogenesis has to be coordinated with leaf growth to enable a switch from heterotrophic to photoautotrophic metabolism. The leaf becomes photosynthetically active at the same time as it shifts from primary to secondary morphogenesis, and chloroplast differentiation is an important regulator of the simultaneous onset of cell expansion and photosynthesis (Andriankaja et al., 2012).
Chloroplasts, like mitochondria, evolved from free-living prokaryotic organisms that entered the eukaryotic cell through endosymbiosis. The division of genetic information between different compartments presents a complex problem to the eukaryotic cell, requiring the coordination of the activities of the different genomes during the process of chloroplast development. Several of the components required for the transcription and translation of the plastid-encoded photosynthesis genes are nuclearly encoded, such as the sigma factors (SIG) of the plastid-encoded RNA polymerase (PEP; Woodson et al., 2013; Chi et al., 2015). In addition, the photosynthetic apparatus is composed of proteins encoded by genes from both the nucleus and the chloroplast. In the photosynthetic electron transport complexes of the thylakoid membrane, for example, the core subunits are encoded by the plastid genome and the peripheral subunits are encoded by the nuclear genome. Thus, it is clear that the initiation of chloroplast development in the light is dependent on the expression of nucleus-encoded components but also that a tight stoichiometric assembly of nucleus-encoded and plastid-encoded proteins is essential for the establishment of photosynthesis (Nelson and Yocum, 2006). To achieve this, there must be communication between the chloroplast and the nucleus through so-called anterograde (nucleus-to-plastid) and retrograde (plastid-to-nucleus) mechanisms (Pogson et al., 2008; Barajas-López et al., 2013a).
The establishment of functional chloroplasts is a complex process involving several cellular compartments. First, chloroplast development is dependent on light, and the initial light signal triggers the activation of the phytochrome and cryptochrome photoreceptors (Waters and Langdale, 2009). These photoreceptors perceive light signals and initiate intracellular signaling pathways involving the proteolytic degradation of signaling components such as phytochrome interaction factors and a large reorganization of the transcriptional program to modulate plant growth and development (Chen et al., 2004). Second, expression of the plastid-encoded photosynthesis genes needs to be initiated, and this induction depends upon the expression and assembly of the nucleus-encoded components, such as the SIGs and polymerase-associated proteins (PAPs), required for the activity of the PEP (Kindgren and Strand, 2015). Third, there are indications from the literature that a hypothetical retrograde signaling mechanism is required for full activation of the nucleus-encoded photosynthesis genes (Woodson et al., 2013). However, it is unclear if this regulatory mechanism is separate from the initial light signal and whether this retrograde signal is positive or negative and, thus, if healthy chloroplasts stimulate or if impaired chloroplasts block nuclear gene expression.
The complexity of leaf development imposes experimental limitations on our ability to study chloroplast biogenesis, restricting the utility of traditional genetic approaches. In planta, chloroplast biogenesis is strongly connected to the development of new tissues and the inhibition of cell elongation; thus, only a few cells at any one moment are in the same state of chloroplast development. In grasses, a spatial gradient of chloroplast development can be observed in a single leaf (Li et al., 2010; Pick et al., 2011), but such developmental gradients in leaves of dicot plants are much harder to isolate. In a single cell-based system, it is possible to observe the synchronous development of many cells in parallel, facilitating the deciphering of the regulatory mechanisms at the level of the individual cell controlling the transition from a proplastid to a functional chloroplast. Thus, to address the limitations of traditional in planta dicot systems, and to develop a well-controlled experimental setup where the individual regulatory checkpoints of chloroplast development could be defined, a pluripotent inducible cell line was generated from Arabidopsis (Arabidopsis thaliana). Following light exposure, this cell line was shown to differentiate into photosynthetically active cells with functional chloroplasts, providing an experimental system with a temporal gradient of chloroplast development. Using this unique cell line from the dicot Arabidopsis in combination with the monocot maize (Zea mays), we could demonstrate that the development from a proplastid to a functional chloroplast, and thereby the establishment of photosynthesis, is dependent on a regulatory mechanism involving two distinct phases. First, light exposure triggers an initial change in gene expression, metabolite profile, chlorophyll accumulation, and plastid structure. Second, a second signal, most likely triggered by activation of the chloroplast, is required for the full transition to a functional chloroplast. A clear checkpoint is associated with the second regulatory phase, as it is dependent on a plastid signal enabling the plant to synchronize the expression of photosynthetic genes from the nuclear and chloroplast genomes during seedling establishment.
An Inducible Cell Line from Arabidopsis Demonstrated the Differentiation of Proplastids to Chloroplasts
A pluripotent inducible cell line was generated from Arabidopsis (Pesquet et al., 2010). This habituated cell line can be propagated in the dark without the addition of hormones or growth factors (Pesquet et al., 2010). We here show that chloroplast development can be triggered on demand in these cells by exposing the culture to light. Green cell culture-based systems that display a photosynthetic capacity similar to the one in planta have been described previously (González-Pérez et al., 2011; Zhang et al., 2011; Hampp et al., 2012). However, in contrast to our cell line, those cell cultures are constitutively green and cannot be grown in the dark, making such systems unusable to investigate the chloroplast developmental process. The cells were grown on Murashige and Skoog (MS) medium supplied with 3% Suc in the dark, and optimal conditions for greening of the cell culture were selected following a screening procedure (methods for subculture, Suc concentrations, light conditions, etc.). A carbon source is required during chloroplast development, and during the deetiolation of dark-grown seedlings the carbon used for the establishment of photosynthesis is taken from the storage in the seed. In a single-cell system, carbon must be provided in the medium. However, to promote source activity (e.g. photosynthesis) rather than sink activity (e.g. growth and storage; Rolland et al., 2002), the amount of sugar in the medium was decreased to 1% prior to exposure to light and the medium was refreshed after 7 d in the light (Fig. 1A). The equilibrated cells at 1% Suc (T0) were exposed to constant light (Fig. 1A). This condition was maintained to allow for sampling at any time point during chloroplast biogenesis and exclude circadian or diurnal fluctuations. The green color of the cell cultures following light exposure also was confirmed by chlorophyll quantification (Fig. 1, B and C). Thus, the chronological progression of chloroplast differentiation in the light was reflected by the increase in chlorophyll content (Fig. 1C).
Analysis using confocal microscopy of the cells at different stages during the light exposure was conducted to investigate the changes in plastid morphology and intracellular position during chloroplast development (Fig. 2A). Combining the xyz-stack images from the confocal microscopy showed that the shape of the plastids in the 5-d cells was poorly defined and the plastids were grouped together (Fig. 2A). Thus, the structure of the 5-d plastids made it difficult to get accurate counts for plastid numbers and positions, but at 7 d, the mean number of chloroplasts per cell reached 42 ± 7. About 61% of the chloroplasts were positioned at the cell cortex, whereas the rest remained clustered around the nucleus (Fig. 2B). In addition, the morphology of the plastids changed into a more defined globular shape of 2.1 ± 0.5 µm in diameter after 7 d in the light (Fig. 2B). After 14 d in the light, the number of chloroplasts per cell remained unchanged (37 ± 7 chloroplasts per cell), but the size of the chloroplasts increased to 4 ± 0.9 µm in diameter compared with the 7-d sample, and nearly 96% of the chloroplasts were now positioned at the cell cortex, as seen in a leaf mesophyll cell (Fig. 2B).
The detailed ultrastructure of the plastids during the greening process was investigated using transmission electron microscopy (Fig. 2C). No prolamellar bodies could be detected in the plastids of the dark-grown samples. Prolamellar bodies are characteristics of etioplasts; thus, the plastids of the dark-grown cell culture were similar to proplastids found in meristematic cells. In the dark, the proplastids displayed an electron-dense aggregate zone that contained large starch deposits and plastoglobuli. Following light exposure for 5 d, an internal structure with small grana of interconnected thylakoids with short intergranal thylakoids appeared in the starch-free plastids (Fig. 2C). After 7 d in the light, the chloroplasts contained a well-developed structure with numerous grana of four to five thylakoids and intergranal thylakoids (Fig. 2C). At 14 d, the thylakoid structure was complete and the size and the morphology of the cell culture chloroplasts were similar to those observed in Arabidopsis and pea (Pisum sativum) plants, where the chloroplasts typically are 5 to 10 µm long and have five to seven thylakoids per grana with intergranal thylakoids (Lopez-Juez and Pyke, 2005; Barajas-López et al., 2013b; Myouga et al., 2013).
Photosynthesis Was Established in the Differentiated Chloroplasts
So far, we have shown that chlorophyll accumulates following light exposure and that the structure and the organization of the chloroplasts in the green cell culture were similar to what was seen in a leaf mesophyll cell. The critical question is whether cultured Arabidopsis cells are capable of performing photosynthesis. First, we looked at the assembly of the photosynthetic protein complexes by analyzing the thylakoid membrane proteins by blue-native PAGE (Fig. 3A; Järvi et al., 2011). Seven major complexes were defined according to Arabidopsis plant thylakoid membrane preparations (Fig. 3A, right lane). We were able to distinguish the known protein complexes following light exposure of the cell culture (Fig. 3A). Most of the photosynthetic protein complexes were already present in the 5-d cells, although at low concentrations, and by 7 d, chlorophyll accumulated in the LHCII trimer (Fig. 3A). Photosystem assembly was completed in 14-d cells, where the protein bands corresponding to the PSII-LHCII supercomplex, PSI-PSII dimer, ATP synthase, PSII monomer, and cytochrome b6f were clearly visible (Fig. 3A). Thus, the protein complex profile of 14-d cells was similar to the profile from leaves of 3-week-old Arabidopsis plants (Fig. 3A). Western-blot analysis confirmed the accumulation of the photosynthetic proteins during the chloroplast differentiation process (Fig. 3B).
To evaluate the activity of photosynthetic electron transport, we measured chlorophyll fluorescence parameters (Fig. 3, C and D). Electron transport was already efficient in 7-d cells, and it increased further in 14-d cells (Fig. 3C). The ratio of variable fluorescence to maximum fluorescence (Fv/Fm), representing the maximum photochemical efficiency of PSII, is 0.73 in the 14-d cells (Fig. 3D). This Fv/Fm was similar to what was frequently reported for 3- to 4-week-old Arabidopsis plants (Hou et al., 2015; Vercruyssen et al., 2015). To confirm photosynthetic activities, oxygen evolution also was determined in 5-, 7-, and 14-d cells (Fig. 3E). By 5 d in the light, the oxygen produced by photosynthesis was already higher than the amounts consumed by respiration. Oxygen evolution increased considerably in the following days. Oxygen evolution in the 7- and 14-d cells was found to be very stable, as the cells could be submitted to many (more than eight) 2-min light/dark cycles without losing oxygen-evolving activity, suggesting that the chloroplasts of the cell culture were very robust. Taken together, these results demonstrate that the cells in this inducible cell culture system behave in many ways like leaf mesophyll cells and can develop functional photosynthetic chloroplasts on demand.
The Establishment of Photosynthesis Was Associated with a Metabolic Change
We investigated the changes in the metabolome during the transition from proplastids to a functional chloroplast and, consequently, the shift from heterotrophic to photoautotrophic metabolism. Relative changes of known metabolites were determined from six biological replicates at each time point. Using this method, 47 metabolites were identified (Supplemental Table S1), and the samples were separated by a principal component analysis (PCA; Fig. 4A). The first two principal components accounted for 63.5% of the total variance of the metabolite data, and the groups differentiated mainly according to the developmental stage (principal component 1). However, two phases during chloroplast development were clearly identified in the metabolome. First, in the response to light, the day-1 to day-4 cells cluster together (constituting phase 1). The metabolome of the 5-d cells was clearly discriminated from the cluster of the day-1 to day-4 cells (principal component 2), suggesting that a critical developmental switch takes place between day 4 and day 5 (initiation of phase 2). Consecutively, 7-, 10-, and 14-d cells were clustered tightly together, suggesting that the cells had switched to photosynthetic metabolism before 7 d. We clustered the metabolite levels (Supplemental Fig. S1) and found that organic acids became less abundant during the transition from proplastid to a functional chloroplast (Fig. 4B). This group was composed mainly of tricarboxylic acid cycle intermediates such as citric acid, succinic acid, fumaric acid, and malic acid (Supplemental Fig. S1). Amino acids cluster in two separate groups, one group that gradually increased and a second that varied in amounts during the developmental process (Supplemental Fig. S1). In the same group as the increasing amino acids, we found other nitrogen-rich metabolites such as allantoin and urea (Fig. 4C; Supplemental Fig. S1). Chloroplast development in this system correlates with large modifications of metabolism, with a depletion of carbon-rich metabolites and an accumulation of nitrogen-rich metabolites. However, at this point, how these metabolite concentrations relate to fluxes through carbon and nitrogen metabolism is difficult to say.
Changes in the Global Expression Profiles of the Nuclear and Plastid Genes Required for the Establishment of Photosynthesis
Changes in nuclear, plastid, and mitochondrial gene expression during the chloroplast development process were determined by RNA sequencing analysis. PCA analysis (Fig. 5) showed that there is a large distance between the clusters of the nuclear genes for day 0 and day 1 (Fig. 5A), indicating that a large shift in gene expression occurred during the first day of light exposure. This agrees with the report that when dark-grown seedlings were exposed to light, as much as one-third of the nuclear genes showed transcript changes (Chen et al., 2010). Another large distance between the clusters was observed between days 4 and 5 (Fig. 5A). After day 5, there was a very small separation of the consecutive clusters for days 7 to 14. This expression pattern of the nuclear genes indicates, similar to what was observed for the metabolome (Fig. 4A), two distinct phases of the regulation of gene expression, with differential expression accentuated between days 0 and 1 and days 4 and 5. Testing for differential expression of the nuclear genes revealed that 3,084 genes were differentially expressed between days 0 and 1 (P < 0.01), whereas 12,411 genes were differentially expressed when day 4 was compared with day 5 (P < 0.01).
The plastid genes also showed a strong change in expression following exposure to light, and the distance between the clusters for day 0 and day 1 was similar to that observed for the nucleus-encoded genes (Fig. 5B). However, the two-phase pattern of gene expression seen for the nuclear genes was not as apparent for the plastid genes, where the separation between the clusters for day 4 and day 5 was less pronounced. The difference in gene expression was most pronounced during the first 5 d and less so from day 5 onward. Expression of the mitochondrially encoded genes changed less in response to light compared with the nucleus- and plastid-encoded genes (Fig. 5C).
Gene Ontology (GO) analyses were performed on the genes that showed a change in expression during the two highly differentiated time points, day 0 versus day 1 (D 0-1) and day 4 versus day 5 (D 4-5). Tables I and II show the top 20 terms within the two ontologies cellular components and biological processes at D 0-1 and D 4-5, respectively. The expression data confirm that chloroplast biogenesis is initiated during the first day of light exposure (D 0-1) and established during the second phase of regulation (D 4-5). Genes categorized under the terms chloroplast (GO:0009507), chloroplast thylakoid membrane (GO:0009535), chloroplast envelope (GO:0009941), and chloroplast stroma (GO:0009570) are found in large numbers among the top-ranked genes for both time points. As many as ∼2,400 differentially expressed genes are tagged with the term chloroplast (GO:0009507) at D 4-5 (Table II).
Regarding the ontology biological processes, the categories response to blue light (GO:0009367) and response to far-red light (GO:0010218) involved in photomorphogenesis were found among the genes changing in expression during the first day of light exposure (D 0-1). The categories photosynthetic electron transport (GO:0009773) and plastid organization (GO:0009657) also were found in the day-0 versus day-1 comparison (Table I). In the day-4 versus day-5 comparison, the terms PSII assembly (GO:0006098), thylakoid membrane organization (GO:0010027), chloroplast relocation (GO:0009902), and photorespiration (GO:0009853) appear in the top 20 GO terms (Table II). Interestingly, the group histone H3-K9 methylation (GO:0051567) including 168 genes appeared in the comparison between the 4- and 5-d samples, suggesting that chromatin modification is involved in the second phase of regulation of the nuclear genes (Table II; Fig. 5A).
A Two-Phase Expression Pattern of Nucleus-Encoded Photosynthesis Components
The expression of genes encoding the components building the photosynthetic machinery was investigated in detail. Heat maps were generated to visualize the expression levels over the time period of chloroplast development and the establishment of photosynthesis (Fig. 6). The expression profiles of the nuclear PSII- and PSI-, cytochrome b6f-, and ATPase-associated genes displayed a very strong induction of expression in response to light when the levels at time 0 were compared with those of the 1-d cells (Fig. 6, A and B). Following the strong light induction, the expression levels were stationary until day 5, when a very strong induction of the expression levels was observed again. This two-phase expression profile also was clear from the PCA plots that showed differential gene expression between days 0 and 1 and days 4 and 5, and this profile appears universal for all the nuclear PSII- and PSI-associated genes. The plastid-encoded photosynthesis-associated genes showed a more gradual increase of expression levels in response to light (Figs. 5B and 6, A and B).
In contrast to the nuclear genes encoding structural components of the photosystems, the nuclear genes encoding photosystem assembly factors (Plöchinger et al., 2016) did not uniformly display the two-phase expression profile. Notably, the expression of ALB3, LPA1, and PSBN was not increased strongly until day 5 (Fig. 6C). The expression profile of the assembly factors correlates with the results from blue-native PAGE (Fig. 3A), where the photosynthetic protein complexes were fully in place first after 7 d in the light and the assembly was completed in 14-d cells. The majority of the nuclear genes encoding the enzymes required for chlorophyll biosynthesis showed the strongest induction in expression levels when day 1 was compared with the control T0 (Fig. 6D), which also coincides with the rapid induction of chlorophyll biosynthesis in response to light (Fig. 1). Interestingly, CAO and HEMA showed a delayed induction of expression compared with the other genes encoding enzymes required for chlorophyll biosynthesis (Fig. 6D).
We also analyzed the expression of nuclear transcription factors known to be involved in chloroplast biogenesis, LONG HYPOCOTYL5 (HY5) and GOLDEN2-LIKE PROTEIN2 (GLK2; Waters et al., 2009; Kobayashi et al., 2012). HY5 activity is correlated directly with the stage of photomorphogenesis (Osterlund et al., 2000). HY5 expression level displayed a strong induction following light exposure in the 1-d sample, and then the expression levels declined (Supplemental Fig. S2). In contrast, GLK2 expression showed an induction first after 4 d in the light (Supplemental Fig. S2). Thus, HY5 and GLK2 showed contrasting expression patterns during the chloroplast developmental process, suggesting different roles. This was also supported by the analysis of hy5 and glk1glk2 mutants (Supplemental Fig. S2). The induction of LHCB expression in response to light was severely impaired in the hy5 mutant but not significantly altered in the glk1glk2 mutant compared with the wild type. In contrast, the more gradual induction of CAO was impaired in glk1glk2 but not in the hy5 mutant (Supplemental Fig. S2).
A Two-Phase Expression Profile for LHCB Was Observed Also in the Spatial Developmental Gradient of a Maize Leaf
In our cell culture system, it is clear that the expression of the key components required for the establishment of photosynthesis occurs in two distinct phases. To test if this regulation also is the case in monocot plants, we used maize leaves, where a gradient of chloroplast development can be observed in a single leaf. Although it is important to emphasize that, in maize, the photosynthetic processes are partitioned between the mesophyll and the bundle sheath chloroplasts, the chloroplast developmental process has been well documented in maize (Li et al., 2010; Pick et al., 2011). The developmental stage of the chloroplasts in the maize mesophyll cells was investigated by transmission electron microscopy at different positions of the leaf (Fig. 7). At the base of the leaf, where the cells are exposed to only limited if any light, the plastids showed a structure indicative of a proplastid, like that observed for the dark-grown Arabidopsis cells (Figs. 2C and 7B). Following the leaf developmental gradient, an internal structure with small grana of interconnected thylakoids was observed 1 cm from the base. At the next sample point, the chloroplasts had a well-developed structure with numerous grana and intergranal thylakoids, and at the tip leaf, the thylakoid structure was complete (Fig. 7B). The expression levels of two LHCB genes were investigated in the maize samples (Supplemental Fig. S3). In the 1-cm sample, a strong induction of LHCB expression compared with the base sample was observed (Fig. 7C). This was similar to what was observed in the 1-d sample from our Arabidopsis cell culture. At the next sample point, 4 cm, a further induction of LHCB expression was observed (Fig. 7C). The expression levels were now stationary, and no further induction was observed at the tip of the leaf (Fig. 7C). Thus, the spatial developmental gradient from the maize leaf showed the same two-phase expression profile as was observed from the temporal developmental gradient in our Arabidopsis cell culture (Fig. 7). In contrast, a gradual increase in plastid PSBA expression along the maize leaf gradient was observed, similar to what was observed in the Arabidopsis cells (Figs. 6A and 7C).
Phase 2 of LHCB Expression Can Be Blocked by the Inhibition of Chloroplast Development
The two-phase expression profile of LHCB genes was observed in two independent greening systems: Arabidopsis cell cultures and maize leaves. In addition, following 3 h of light exposure of etiolated seedlings, a strong induction of LHCB expression was observed. The expression levels were then stationary for the first 12 h of light exposure, but between 12 and 24 h, a second significant induction in gene expression was observed, and between 24 and 48 h of light exposure, no further induction was observed (Supplemental Fig. S4). The collected data suggest that the second phase of induction of the nucleus-encoded photosynthesis genes is linked to a certain developmental stage and/or activation of the chloroplasts. To test if the induction of gene expression is linked to the status of the chloroplast, we used inhibitory agents to block proper chloroplast development. We exposed the cells to high Suc concentrations (Supplemental Fig. S5). Chloroplast development was blocked by high Suc concentrations, and the cells were then unable to perform photosynthesis (Supplemental Fig. S5). As an alternative condition, we used lincomycin, an inhibitor of plastid protein translation (Fig. 8). The initial induction of the LHCB expression triggered by light exposure was not affected following the lincomycin or high Suc concentrations (Fig. 8; Supplemental Fig. S5). However, the second phase of induction was absent following the Suc and lincomycin treatments when chloroplast development was blocked. Thus, phase 2 of the regulatory mechanism controlling LHCB expression was shown to be distinct from the initial light response and clearly linked to proper chloroplast development, as was shown both in cell cultures and Arabidopsis seedlings (Fig. 8; Supplemental Fig. S5).
Mathematical Calculations Suggest a Feedback-Controlled Coordination of Nuclear and Plastid Gene Expression
The initial light response is mediated via nuclear components, but our results (Figs. 6–8) strongly suggest that a retrograde signaling mechanism is required for the full expression of photosynthesis-associated nuclear genes (PhANGs). To test this conclusion, we mathematically assessed if the nuclear and plastid genomes are connected via positive feedback (Mitrophanov and Groisman, 2008). Positive feedback has been demonstrated to contribute to the efficiency of transcriptional regulatory systems (Mitrophanov and Groisman, 2008). To convert our experimental data to mathematical parameters, the expression of PSII and PSI components was recalculated to a geometric mean (Fig. 9). Nuclear gene expression (y axis) displayed a large dynamic range, with two distinct phases, where low and high expression clustered together. The calculations displayed a linear coordination of expression from the nucleus and the plastids for low expression levels (Fig. 9, A and B). This linear coordination could be explained by the fact that the nucleus-encoded SIGs and PAPs are required for PEP activity and the expression of PSII and PSI components in the plastid. Following this initial linear phase, a dramatic increase in expression levels occurred and the nuclear expression levels were elevated to a second phase. However, when chloroplast development was inhibited by high Suc or lincomycin treatment, nuclear gene expression followed the initial linear response of the two genomes (Fig. 9C, black circles) but did not enter the second high-expression phase. This suggests that the second phase is due to a positive retrograde feedback, linked to plastid activities, regulating nuclear gene expression. Mathematically, when two components display an initial linear response for small values with an increasing nonlinear response and a distinct jump to a second phase, it is demonstrated that the two components are mutually connected through a positive feedback mechanism (Supplemental Data S1). Thus, the calculations support our conclusions that the two components, nucleus and chloroplast, are coordinated by positive feedback during chloroplast development and that the activation of chloroplast transcription is the trigger for the feedback signal required for the full expression of PhANGs.
Using a single cell culture from Arabidopsis, we could show that the establishment of photosynthesis through the development from proplastids to functional chloroplasts occurs in two distinct phases (Fig. 10). During the initial phase, light exposure triggers a significant change in gene expression (Fig. 5) and the metabolite profile changes in response to light (Fig. 4). As many as 3,084 genes were differentially expressed between day 0 and day 1, and genes within the categories response to blue light (GO:0009367) and response to far-red light (GO:0010218) were highly represented among those genes (Table I), confirming the important role of the photoreceptors during the early light response (Strasser et al., 2010). Following the initial response to light exposure, the expression levels remained rather stationary until day 5, when the second phase of the developmental process was triggered. During phase 2, the final transition to photosynthetically functional chloroplasts occurs, and this transition requires a major reorganization of cellular metabolic activity. Such a complete reorganization of the metabolism will naturally require the expression of novel components, and as many as 12,411 genes were differentially expressed when day 4 was compared with day 5 (P < 0.01; Fig. 5). The GO enrichment also showed that a more diverse set of genes was triggered in the second expression phase, although a high percentage of the genes would fall within the category chloroplast (GO:0009507; Table II). Interestingly, the group histone H3-K9 methylation including 168 genes suggested that chromatin modification could be involved in the second phase of regulation of the nuclear genes (Table II; Fig. 5A). In mammalian cells, it was shown that changes to intracellular metabolism alter the expression of specific histone methyltransferases and acetyltransferases, conferring widespread variations in epigenetic patterns (Keating and El-Osta, 2015). Thus, the large number of genes changing in expression between day 4 and day 5 also would argue for a regulation of gene expression at a higher level of organization. Associated with the massive change in gene expression during phase 2 was a change in the metabolome, supporting a critical metabolic shift between day 4 and day 5 (Fig. 4).
In contrast to the nucleus-encoded genes, the plastid and mitochondrial genes did not show the same two-phase expression profile, and the increase in photosynthetic gene expression was more gradual in response to light. PEP represents the major transcription machinery in mature chloroplasts, and over 80% of all primary plastid transcripts are transcribed by PEP (Zhelyazkova et al., 2012). The initiation of chloroplast development in the light and the activation of the photosynthetic reactions are believed to be accompanied by a repression of NEP activity and an increase of PEP-mediated plastid transcription (Liere et al., 2011; Börner et al., 2015). However, the mechanisms underlying this change in major RNA polymerase activity and the division of labor between NEP and PEP in the chloroplast are unknown (Zhelyazkova et al., 2012). The transcript levels for the plastid-encoded photosynthesis components were relatively high compared with the nucleus-encoded components at T0, suggesting that the transcription of plastid-encoded photosynthesis genes occurs to some degree already in the proplastids in the dark. Possibly, this indicates the NEP-driven expression of those components already in the dark.
The second phase of nuclear gene expression coincided with the appearance of the first chloroplast-like structures. At day 5, an internal structure with interconnected thylakoids appeared in the plastids (Fig. 2), and some photosynthetic activity could be detected (Fig. 3). During phase 2, the photosynthetic complexes are assembled and finalized. More than 40 proteins are associated with PSII, either stably or transiently (Järvi et al., 2011). Due to such complexity, the photosystem apparatus must be assembled in an orderly manner, ensuring that the components are inserted in the correct sequence. It appears unlikely that this is controlled by the order in which different components are expressed, as the data showed that they are all expressed simultaneously following illumination. The photosystem assembly factors, such as SRP54, ALB3, LPA1, and LPA2, play a key role in the assembly of a functional PSII and PSI, and full expression of these factors was not observed until day 5 (Fig. 6C). The expression profile of the assembly factors correlates with the results from blue-native PAGE (Fig. 3A), where the photosynthetic protein complexes only appeared after 7 d in the light. The assembly of PSII and PSI was completed in 14-d cells, when all the photosynthetic protein complexes, such as the PSII-LHCII supercomplex, PSI-PSII dimer, ATP synthase, PSII monomer, and cytochrome b6f, were clearly visible by green-native PAGE (Fig. 3A).
During chloroplast formation, distinct morphological changes to the plastids were observed, including a gradual change in chloroplast shape from elongated tubes to globular organelles (Fig. 2). The morphological changes were accompanied by a clear positional shift from the nuclear vicinity to the cellular cortex (Fig. 2). Thus, we have demonstrated that the shape and the cellular position of the plastids are highly dynamic during the transition from proplastid to functional chloroplast. A critical developmental switch occurred between day 4 and day 5, as indicated by the massive change in gene expression and the change in the metabolome. At this time point, the plastids appeared clustered around the nucleus (Fig. 2A), possibly to facilitate the information flow from the nucleus to the chloroplasts. As the development into a functional chloroplast proceeded, the plastids moved toward the plasma membrane. In the 7-d cells, some plastids appeared near the cell cortex, whereas following 14 d in the light, the chloroplasts were clearly separated from each other and positioned at the cell cortex, as seen in leaf mesophyll cells (Fig. 2A). The final position of the chloroplasts at the cell cortex also was associated with fully functional chloroplasts and the establishment of true photosynthetic activity. Thus, the development of functional chloroplast also is associated with major cytological changes, where the cellular position of the plastids is very dynamic. Chloroplasts were shown to move from the cell cortex to the nucleus upon illumination as a mechanism of photoavoidance in the centric diatom Pleurosira leavis (Furukawa et al., 1998), but the movement of plastid during chloroplast development, to our knowledge, has not been demonstrated previously.
The second phase of expression of the nucleus-encoded photosynthesis genes was clearly linked to the activity of the chloroplast. Correlated with the large change in nuclear gene expression between day 4 and day 5 was the establishment of a significant expression of the plastid-encoded photosynthesis genes (Figs. 6 and 9). When the expression profile of the temporal chloroplast differentiation gradient in our Arabidopsis cell line was compared with the gradient described in a single leaf of maize, the spatial developmental gradient from the maize leaf showed the same two-phase expression profile (Fig. 7). The maize leaves contain the two cell types, mesophyll and bundle sheath cells, with different roles in the photosynthetic process; thus, the samples for the expression data come from a mix of the two cell types. However, the second induction of LHCB expression was correlated with the formation of plastid structures in the mesophyll cells and the expression of the plastid-encoded photosynthesis genes (Fig. 7). In addition, published expression profiles from maize showed that samples from the midpoint of the leaves showed a strong induction of photosynthesis genes whereas the genes encoding the enzymes in tetrapyrrole biosynthesis were expressed closer to the base of the leaf, similar to the profile detected in the Arabidopsis cell line (Fig. 6; Li et al., 2010; Pick et al., 2011). A good correlation between expression levels and protein levels of photosynthetic components has also been demonstrated (Majeran et al., 2010). Thus, gene expression profiles of those different categories of photosynthesis-related genes were linked to the same developmental stages of the plastids in the two different systems representing dicots and monocots, respectively (Figs. 6 and 7).
The initial induction of LHCB expression triggered by light exposure was not affected by the lincomycin treatment or the high Suc concentrations (Figs. 8 and 9; Supplemental Fig. S5). In contrast, the second phase of induction was absent when chloroplast development was blocked. Thus, the mechanism behind phase 2 of PhANG expression is separate from the initial light signal (Fig. 8). The second phase is most likely induced by a retrograde signal dependent on normal chloroplast development (Fig. 9). Our mathematical calculations suggest that the nucleus and the plastids are mutually connected by a positive feedback mechanism (Fig. 9). Once PEP is activated and the expression of the plastid-encoded photosynthesis genes is induced, we postulate that a positive plastid signal is generated through a retrograde mechanism. The calculations and the expression profiles show that this retrograde signal is most likely linked to the transcription of plastid-encoded photosynthesis genes. The status of the PEP complex links the functional state of the chloroplast to the nucleus, enabling the plant to synchronize the expression of photosynthetic genes from the nuclear and chloroplast genomes during seedling establishment. The plastid signals operating during chloroplast biogenesis have not yet been discovered, but the lack of PhANG expression when chloroplast development is blocked by mutations or chemical treatments has usually been interpreted as being due to a repressive signal emanating from damaged or abnormal plastids. However, the possibility was recently presented in which lincomycin would either (1) disrupt a positive plastid-emitted signal, which acts in a GENOME UNCOUPLED1 (GUN1)-regulated manner, or (2) induce a negative plastid-emitted signal, which acts to repress nuclear transcription in a GUN1-mediated manner (Martín et al., 2016). Our results conclude that PhANG expression is dependent on a positive signal from healthy developing plastids. In this new understanding of the coupling of nuclear and plastid gene expression, the gun mutations could be interpreted as allowing a positive signal to proceed that would normally have been shut down. In this case, the mutated components would be expected to be part of a gating machinery that controls whether the signal is produced. This new interpretation may help us reinterpret puzzling old data or generate new hypotheses to test.
Cell Culture, Growth Conditions, and Cell Sampling
Arabidopsis (Arabidopsis thaliana) Col-0 cell lines were grown in MS medium supplied with 3% (w/v) Suc, pH 5.7, in the dark at 25°C and shaken at 140 rpm (Pesquet et al., 2010). Cells were subcultured weekly by a 1:10 dilution. For all experiments, 7-d-old cells from dark conditions were subcultured in a 1:10 ratio in MS medium with 1% (w/v) Suc, equilibrated, and placed in a growth cabinet under continuous light (150 µmol photons m−2 s−1) and constant rotary agitation. After 7 d in the light, cells were pelleted without centrifugation and the medium was replaced. Cells were placed back in the light until 14 d for the final sample. Cells were collected from the culture by filtration, immediately frozen in liquid nitrogen, and ground in a mortar. For metabolomics analysis, a washing step with ice-cold distilled sterile water of the filtered cells was added before freezing the samples. The oxygen evolution rate and chlorophyll fluorescence parameters were determined with 1- and 2-mL aliquots of cell culture, respectively.
Plant Growth Conditions and Tissue Collection
All experiments were performed with Arabidopsis ecotype Col-0. The glk1glk2 double mutant and hy5-1 mutant lines were described elsewhere (Fitter et al., 2002; Maxwell et al., 2003; Kleine et al., 2007). For the etiolated seedlings, seeds were surface sterilized and cultured on phytagel-solidified (2.7 g L−1; Sigma-Aldrich) MS medium (2.2 g L−1; Duchefa) including Suc (10 g L−1; VWR) and MES buffer (0.5 g L−1; Sigma-Aldrich), pH 5.7. The MS plates were covered with aluminum foil and kept for 3 d at 4°C in darkness and then moved into continuous white light (150 µmol m−2 s−1) for 3 h at 22°C to potentiate germination. MS plates were kept in total darkness at 22°C for 5 d. After 5 d, the etiolated seedlings were exposed in continuous white light (550 µmol m−2 s−1) and 22°C. For lincomycin treatment, the etiolated seedlings were transferred onto MS plates containing 1 mm lincomycin (Sigma-Aldrich) or distilled, deionized water as a mock treatment and then exposed to continuous white light (150 µmol m−2 s−1) and 22°C.
For maize (Zea mays) samples, tissue was collected from the third leaf of 9-d-old maize B73 seedlings according to Li et al. (2010). Briefly, seeds were sown in the soil directly and grown under light of 150 µmol m−2 s−1, 12/12-h light/dark, 31°C light/22°C dark, and 50% relative humidity. Four different segments from each leaf were collected: basal (1 cm above the third leaf ligule), transitional (1 cm below the second leaf ligule), maturing (4 cm above the second leaf ligule), and mature (1 cm below the third leaf tip).
Chlorophyll Analysis
Chlorophyll was extracted by adding 1 mL of buffered acetone (80% acetone and 0.2 m Tris-HCl, pH 7) to 80 mg fresh weight of cells. Samples were incubated overnight at 4°C and centrifuged for 10 min at 15,000g. Chlorophyll content was measured and expressed according to Porra et al. (1989).
Confocal and Transmission Electron Microscopy
Analysis of chlorophyll autofluorescence was performed by confocal laser scanning microscopy (Zeiss LSM 780). The cell wall was stained by incubating samples with Calcofluor White (0.002% final concentration) for 5 min. Cells were scanned sequentially to prevent any cross talk between fluorescence channels. Confocal 0.37-µm-thick optical sections of bright-field, Calcofluor White staining, and chlorophyll autofluorescence signals were combined for a three-dimensional reconstruction of cells using ImageJ. Chloroplast density, diameter, and cortical positioning were determined using ImageJ. As the chlorophyll intensity is low in 5-d cell samples compared with 7- and 14-d cell samples, the level of chlorophyll intensity was adjusted to correctly visualize the plastids in the 5-d cell sample. For transmission electron microscopy, the samples were fixed using 2.5% (v/v) glutaraldehyde in 0.1 m cacodylate buffer overnight at 4°C. After washing three times in buffer, the specimens were postfixed with 1% (v/v) osmium tetroxide in the medium buffer for 1 h and washed twice in distilled water. Samples were dehydrated with 50%, 70%, 95%, and 100% ethanol and infiltrated and embedded in Spurr's resin. Using a Diatome diamond knife on a Leica EM UC7 device, thin sections (60–90 nm) were collected onto copper grids, treated with 5% uranyl acetate in water for 20 min, followed by Sato's lead staining for 5 min. Sections were examined in a JEOL 1230 transmission electron microscope, and digital images were captured using a Gatan MSC 600CW camera.
Isolation of Thylakoid Protein Complexes and Blue-Native PAGE
Thylakoid membrane purification was done according to Hall et al. (2011) with modifications. Briefly, cells were pelleted by centrifugation (200g, 2 min), incubated with cellulase (1%, w/v) and macerozyme (0.2%, w/v), and shaken in the dark for 4 h to digest the cell wall. After homogenization, successive centrifugation, and washing steps, thylakoid membranes were resuspended in a thylakoid wash buffer (Hall et al., 2011). Protein quantification was done with the Pierce BCA Protein Assay Kit (Thermo Scientific) after precipitation of the proteins in chlorophyll extraction buffer and resuspension of the pellet in 160 mm Tris-HCl and 2% SDS. A total of 50 µg of protein complexes from isolated thylakoids was diluted in 25 mm Bis-Tris-HCl, pH 7, and 2% glycerol and solubilized with 3% β-dodecylmaltoside (Sigma-Aldrich) for 10 min at 4°C in the dark. Unsolubilized material was removed by centrifugation at 18,000g for 20 min. A total of 35 μg of protein was loaded with BN loading buffer on a 4-12% Bis-Tris gel (NuPAGE Novex 1.0 mm; Invitrogen).
Protein Gel Electrophoresis and Immunoblotting
Total proteins were extracted from 100 mg fresh weight of cells with 200 µL of buffer (65 mm Tris-HCl, pH 6.8, 10% glycerol, 3% SDS, 0.005% Bromophenol Blue, and 5% β-mercaptoethanol), heated at 95°C for 5 min, and spun down for 10 min at 14,000 rpm. A total of 15 µL of protein extract was loaded, and proteins were separated on a 10% acrylamide gel for SDS-PAGE analysis and then transferred on a nitrocellulose membrane for western-blot analysis. Proteins LHCB1, LHCB2, psaC, psbD, and RBCL were detected with primary rabbit antibodies at dilutions of 1:5,000 (LHCB1 and LHCB2), 1:100 (psaC), 1:2,000 (psbD), and 1:2,500 (RBCL; Agrisera) and a secondary donkey anti-rabbit IgG antibody conjugated to horseradish peroxidase (1:50,000). For α-tubulin detection, a primary mouse antibody (1:1,000) and a secondary goat anti-mouse IgG antibody conjugated to horseradish peroxidase (1:10,000) were used. Luminescence was detected using the ECL Prime Western Blotting Detection Reagent (Amersham).
Oxygen Evolution Analysis and Chlorophyll Fluorescence
Oxygen evolution was measured using a 1-mL cell suspension and an oxygen electrode (Hansatech) at 20°C (no external electron acceptors were added). Samples were dark incubated on the electrode with slow steering for 2 min and then illuminated with saturating white light (2,000 µmol photons m−2 s−1) for 2 min. Oxygen evolution was recorded and evaluated by Oxygraph version 1.15 (Hansatech), and the rates were converted into nanomoles of oxygen produced per minute and standardized to total fresh weight of cells. Chlorophyll fluorescence was measured using a Dual-PAM-100 (Walz) on a 2-mL aliquot of cell culture in a glass cuvette under agitation. Before each measurement, samples were dark adapted for 20 min. The F0 (minimum fluorescence yield) was measured under weak modulated measuring light (9 µmol photons m−2 s−1) and the Fm (maximum fluorescence yield) was measured by applying a saturating pulse of white light (3,000 µmol photons m−2 s−1 for 0.6 s). The actinic light was 125 µmol photons m−2 s−1. Fv/Fm was calculated from the ratio (Fm – F0)/Fm.
Metabolite Analysis
A total of 30 mg of frozen cell powder from six independent cell cultures (biological replicates) was submitted to metabolite analysis according to Kusano et al. (2011), except for the statistical analysis. Metabolites present in the UPSC library were automatically investigated in the chromatograms, and corresponding peaks were integrated and quantified. Obtained peaks were subsequently confirmed by manual curation against the UPSC library and the Golm library (Schauer et al., 2005). A matrix was then generated containing the peak areas (normalized using the internal standards and the fresh weight) and the samples and then was used for statistical analysis performed with SIMCA13.0.3 software.
RNA Isolation and RNA Sequencing
Total RNA was isolated using the mirVana miRNA Isolation Kit (Ambion) according to the manufacturer's instructions. RNA was quantified with a Nanodrop ND-100 spectrophotometer, and RNA quality was checked by electrophoresis on a 1% agarose gel. RNA samples were ethanol precipitated and diluted to 500 ng μL−1. RNA quality was assessed using an Agilent 2100 bioanalyzer (Agilent Technologies), and the RNA was quantified with a Qubit 2.0 fluorometer (Thermo Fisher Scientific), both according to the manufacturer's instructions.
RNA sequencing libraries were prepared using the TruSeq Stranded Total RNA with Ribo-Zero Plant Kit (Illumina), according to the manufacturer's instructions, using half-reactions and 200 ng of RNA. Successful rRNA depletion was verified by electrophoresis using the High Sensitivity RNA ScreenTape System on the Agilent 2200 TapeStation (Agilent Technologies), according to the manufacturer's instructions. Enrichment of cDNA fragments was performed using 11 cycles generating cDNA fragments of ∼270 bp. Prior to sequencing, single-molecule DNA templates were bridge amplified on the cBot (Illumina) to form clonal clusters inside the flow cell using the TruSeq SR Cluster Kit v3-cBot-HS (Illumina), according to the manufacturer's instructions. Single-read sequencing was carried out for 51 cycles on the HiSeq 1500 (Illumina) using the TruSeq SBS Kit v3-HS (Illumina), following the manufacturer's instructions. An average cluster density of ∼850 K mm−2 was recorded.
Computational Analysis
Raw read data were converted to fastq files using the Casava program and assessed for quality using the program FastQC version 0.11.3 (www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were then filtered using the program Trimmomatic version 0.33 (Bolger et al., 2014), removing the Illumina adapters and reads with quality below 20. Raw read count numbers per gene were obtained via pseudoalignment using the Kallisto program (Bray et al., 2015). TAIR10 genome and accompanying gene annotation file were used as references (www.arabidopsis.org). A correction was first made to the YCF3 gene annotation (ATCG00360) in accordance with de Longevialle et al. (2008). Raw counts produced by Kallisto were processed using a custom Java script that took the maximum isoform count for each gene and rounded it to an integer value for compatibility with downstream analysis tools. The resulting single matrix of raw count data was split into three separate count matrices based on the encoding compartment (nucleus, plastid, and mitochondrion). Each of these data sets was normalized separately using the DESeq2 package (Love et al., 2014). This was performed using the DESeq likelihood ratio test. PCA was carried out within DESeq2 using a variance-stabilizing transformation on count data. The reads from RNA sequencing have been uploaded to the European Nucleotide Archive database (accession no. E-MTAB-5777).
GO Term Enrichment Analysis
Differential gene expression results of nuclear genes for D 0-1 and D 4-5 were obtained from normalized count data in DESeq2 using a Wald test with a log-fold change threshold of 1 and an adjusted P value cutoff of less than 0.01. Annotation of differentially expressed genes was provided by the R package GO.db (bioconductor.org/packages/GO.db). Background genes were determined using the Manhattan method of genefinder in the R package Genefilter (bioconductor.org/packages/genefilter). Top GO terms were compiled with the R package topGO (Alexa and Rahnenfuhrer, 2010) using the Fisher test statistic.
Gene Expression Analyses
Total RNA was extracted from Arabidopsis seedlings or segments of maize leaf using the E.Z.N.A.Plant RNA Kit (VWR). The extracted total RNA was quantified with an ND-1000 spectrophotometer (NanoDrop Technologies). One microgram of total RNA was used as a template for reverse transcription with the iScript cDNA Synthesis Kit (Bio-Rad) before the possible genomic DNA was eliminated with DNase I (Thermo Fisher Scientific). Equal amounts of first-strand cDNAs were used as templates for real-time PCR amplification using the following primer combinations (Supplemental Table S2): qLHCB1.1_F/qLHCB1.1_R, qLHCB2.4_F/qLHCB2.4_R, qCAO_F/qCAO_R, qZm2G351977_F/qZm2G351977_R, qZm2G120619_F/qZM2G120619_R, and qZMPSBA_F/qZmPSBA_R. For Arabidopsis, PROTEIN PHOSPHATASE2A SUBUNIT A3 (PP2A; AT1G13320) was amplified using the primer combination qPP2A_F/qPP2A_R. For maize, ZmUBI (qGRMZM2G102421) was amplified using the primer combination qZm2G102421_F/qZm2G102421_R. Quantitative real-time PCR was performed using iQ SYBR Green Supermix (Bio-Rad) with a CFX96 Real-Time System (Bio-Rad) quantitative PCR machine. LHCB1.1, LHCB2.4, and CAO transcript levels were quantified in relation to Arabidopsis PP2A levels, and GRMZM2G351977 and GRMZM2G120619 transcript levels were quantified in relation to ZmUBI levels.
Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. Heat map and cluster representation of the metabolite profiles in the different samples during chloroplast development.
Supplemental Figure S2. Gene expression of HY5, GLK2, LHCB1.1, and CAO in Arabidopsis cell culture and seedlings.
Supplemental Figure S3. Phylogenetic analysis of the maize family genes, showing the phylogenetic relationships among LHCBs and PSBAs in maize and Arabidopsis.
Supplemental Figure S4. Deetiolation of Arabidopsis seedlings.
Supplemental Figure S5. High Suc concentration inhibits chloroplast development.
Supplemental Table S1. List of identified metabolites with their normalized peak areas in every sample.
Supplemental Table S2. List of primers used for the RT-PCR analysis.
Supplemental Data S1. Theory section with details of the mathematical calculations.
We thank Dr. Jane Langdale for the glk1glk2 seeds.
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: Åsa Strand (asa.strand{at}umu.se).
Å.S., C.D., W.P.S., J.H., and I.S. designed the experiments; C.D., X.J., T.C.H., S.K.T., T.D., W.P.S., A.G., and E.P. performed the experiments; all authors contributed to data analysis, writing of the article, and reviewed and approved the final version of the article.
1 This work was supported by grants from the Swedish Research Council (to Å.S. and E.P.) and Solar Fuels/Artificial Leaf grants (to Å.S. and W.P.S.), the Kempe Foundation (to J.H. and T.D.), the Carl Tryggers Foundation (to W.P.S.), and the Australian Research Council (grant no. CE140100008 to I.S.).
2 Current address: Molecular Plant Biology, Department of Biochemistry, University of Turku, FI-20520 Turku, Finland.
- Murashige and Skoog
- principal component analysis
- Gene Ontology
- Col-0
- Columbia-0
- Received April 3, 2017.
- Accepted June 9, 2017.
- Published June 16, 2017.


text:
Finance Minister Lim Guan Eng has warned telcos to stop charging consumers 6% sales and service tax (SST) to consumers for prepaid cards. According to multiple media reports, the government wants to ensure that consumers are getting the full RM10 amount when they top up their prepaid cards.
He was quoted the New Straits Times saying that a directive has been issued to telcos and they are expected to comply with it. Media reports also quote Lim saying the government will interfere if the directive is disobeyed.
Only one-third of goods are subjected to SST and among the list of service providers impacted include night clubs, betting and gaming providers, airline operators and IT service providers. Those with revenue exceeding RM1.5 million must pay SST.
Meanwhile, he also criticised former Prime Minister Datuk Seri Najib Tun Razak (pictured) who complained about being charged SST of RM3.80 for steamed buns.
Najib had taken to Twitter to complain about being charged SST at Pau Yik Mun restaurant, which he claimed was a "mid-sized" store. The goods and services tax was replaced with the SST on 1 September, after the parliament passed the Sales Tax Bill 2018 and Service Tax Bill 2018. The SST comprises 10% for sales tax and 6% for services.
Berhenti di kedai Pau Yik Mun Tg Malim untuk membeli Pau . Jelas SST dikenakan walaupun kedai ini sederhana kecil ! Kedai Pau pun kena SST ? pic.twitter.com/68s5UT14TB
— Mohd Najib Tun Razak (@NajibRazak) September 9, 2018
According to Bernama, Lim said he was "astonished" that Najib did not grumble about the GST when it was implemented previously, neither did he listen to the complaints of Malaysians. He added that the 6% GST imposed previously was "far worse", and that the government is "being quite flexible compared to before".

subset: commoncrawl


text:
There is such a configuration on my Nginx 1.2.1 server:
server { listen 80 default_server; listen [::]:80 default_server ipv6only=on; root /var/www; index index.php index.html; autoindex on; autoindex_localtime on; try_files $uri $uri/ =404; location ~ ^/~([^/]+)(/.*)?\.php(/.*)?$ { alias /home/$1/public_html$2.php; fastcgi_pass unix:/var/run/php5-fpm.sock; fastcgi_index index.php; include fastcgi_params; fastcgi_param SCRIPT_FILENAME /home/$1/public_html$2.php; fastcgi_param PATH_INFO $3; } location ~ ^/~([^/]+)(/.*)?$ { alias /home/$1/public_html$2; } }
It aims to map /~username/ to /home/username/public_html/ so that users on my server can access and manage their own files.
However, this brought a problem. If one tried to access a non-exist .php file, such as /~username/asdfasdf.php, php-fpm would complain about "Invalid Argument" instead of displaying the "404 Not Found" page of Nginx.
I have browsed Nginx Wiki and found:
Note that there is a longstanding bug that alias and try_files don't work together.
But I would never give up!
I observed the environment variables that Nginx passed to PHP and found that $_SERVER["DOCUMENT_ROOT"] became the same value as $_SERVER["SCRIPT_FILENAME"] instead of /var/www.
try_files directive tries files from $document_root. For example, try_files $uri =404; tests the existence of the file named $document_root$uri.
It is clear after I noticed this. The workaround is just append try_files "" =404; after the line of alias.


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|Bomi Park, illus. by Bomi Park
|Chronicle Books, 2016.
5 stars
|Keywords: 0-3 ages bomi-park first-snow
First Snow
by Bomi Park, illus. by Bomi Park
Bomi Park's little picture book, First Snow, captures the magic of the first snow fall. Illustrated in gorgeous black and white charcoal. The only hint of color is the little girl's red scarf.
On the first page, the snow begins to fall, and the narrator says, "Shhhh, listen…do you hear something?" The little girl crawls to the window to see snow falling against the window pane.
"Quick! Boots, coat, scarf, hat."
And while the world is sleeping, the child steps outside, marveling at her footprints in the snow. She is joined by a small dog, and other animals along the way. She rolls a small snowball until it becomes a big one. And when she makes it on the other side of the woods filled with snow covered evergreen trees, there is a field full of children who also have rolled their snowballs silently through the night. One by one they join together to create snowmen! There are a couple pages that have no text on them, and those illustrations evoke the silent fall of snow. In particular, there is a great full-page illustration of the children and their accompanying snowmen floating into the sky. A child's first snow is truly magical, and Bomi Park has captured a slice of that magic in her picture book.


text:
Hurricane Kiko Forecast Discussion Number 12
2019-09-15 10:55:11| National Hurricane Center (East Pacific)
Issued at 200 AM PDT Sun Sep 15 2019 000 WTPZ43 KNHC 150855 TCDEP3 Hurricane Kiko Discussion Number 12 NWS National Hurricane Center Miami FL EP132019 200 AM PDT Sun Sep 15 2019 Kiko has continued to strengthen overnight and is now characterized by a cold ring of convection with cloud top temperatures below -70 degrees Celsius surrounding a clearing, well-defined eye. Both subjective and objective satellite intensity estimates have increased, and a blend of these estimates indicate an initial intensity of 110 kt. The improved appearance and structure of Kiko over the past several hours suggests that some additional strengthening is possible today, and the official forecast makes Kiko a category 4 hurricane by this afternoon. The intensity forecast beyond that time becomes challenging. Dynamical and statistical guidance unanimously weaken Kiko over the next few days. However, other than a slight increase in northeasterly shear indicated in SHIPS guidance, there are no other environmental indicators that stand out as a good reason for Kiko to weaken as quickly as shown by that guidance. It is also interesting to note that there is significant model spread in the forecast position of Kiko by 72 hours. Some of the guidance places Kiko over cool SSTs by day 3, while some solutions keep Kiko over warmer water. The official foreast, which is similar to the previous one, does indicate some weakening through 72 hours, but at a slower rate than the majority of the guidance since the latest forecast has trended southward over warm water. After 72 hours, increasing southwesterly shear from an upper-level trough to the northwest of Kiko should cause steady weakening after that time. Kiko is moving just south of due west, or 265/7 kt. A mid level ridge to the north of the hurricane should keep it generally on a west or west-northwestward track for the next couple of days. As mentioned previously, the models diverge by 72 hours as they differ in how they handle a mid to upper-level trough approaching California from the west. The northernmost solutions produce a break in the ridge with a sharper trough. The southern solutions keep the ridge in tact. Based on the continuation of a slight southward trend in the guidance, and the southward bias in the actual track of the cyclone recently, I am placing a little more weight on the southern solutions. However, given the large spread beyond 72 hours, the track confidence during that time is low. FORECAST POSITIONS AND MAX WINDS INIT 15/0900Z 16.9N 120.7W 110 KT 125 MPH 12H 15/1800Z 16.9N 121.8W 115 KT 130 MPH 24H 16/0600Z 17.2N 122.9W 105 KT 120 MPH 36H 16/1800Z 17.5N 124.0W 100 KT 115 MPH 48H 17/0600Z 17.6N 124.9W 95 KT 110 MPH 72H 18/0600Z 17.8N 126.6W 80 KT 90 MPH 96H 19/0600Z 18.1N 128.4W 65 KT 75 MPH 120H 20/0600Z 18.5N 131.0W 55 KT 65 MPH $$ Forecaster Latto
Tags: number
Category:Transportation and Logistics


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Forwarding Mail
Students who leave Dartmouth or the Hanover campus for a term must forward their mail to their new address.
Please change your address in Banner. Please activate the forwarding address in Banner. When boxes are closed for a term, the Hinman Mail personnel use the Banner system to acquire the forwarding address.
Upon graduation, your mail will be forwarded for 90 days. The United States Postal Service will forward UNOPENED first class mail to anywhere in the world without requiring additional postage.
Magazines and standard mail cannot be forwarded. Please notify your magazine subscribers of a change in address to continue uninterrupted receipt of publications.


text:
W. Chestnutt Charles eBooks
eBooks found: 1
The Marrow of Tradition
W. Chestnutt Charles
ReadHowYouWant, October 2009
Format: ePub
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text:
Playful Creativity Inspired by Art Games and the Surrealist Artists
Start with play!
Learning begins with personal free play that is open to possibility, open to discovery, and open to unconscious connections. We let down our guard, surrender to imagination, and rediscover memories.
There are many ways to start art activities, but the key is the playful surrender. Random possibilities, freedom and lack of judgement encourage possibilities.
For example, textured or altered surfaces, seeing images in ink blots, coffee stains, natural wood grain, watercolour spills, smudged smoke or charcoal, etc., all offer possibilities to trigger imaginative images. We can set up the materials, start question games, tell a story or play some music and then possibly set a timer so children just start without overthinking.
Today we will start with Exquisite Characters and Character Collages, as inspired by games played in coffee shops by the Surrealist artists a century ago.
will start our process today
Video about Surrealism for kids. The focus is on Dali but Surrealist strategies are also explained in ways that are easy to understand.
Slideshow with background information about Surrealism, including artists.
RESOURCE with examples of SURREALIST GAMES FOR KIDS by Cindy Ingram, from her art education blog "The Art Curator for Kids."
Student Digital Collage Examples from OKM
Combining collage with drawing
One-eyed monsters, zombies and other creatures created from cut-out found image parts, completed with drawing or painting.
Sketchbook drawing example from Madeleine de Kemp from The Netherlands, inspired by Lab number 26, exercise from the book DRAWING LAB by Carla Sonheim: LINK WITH SAMPLES POSTED TO FLICKR
Next week our topic is IMAGE DEVELOPMENT.
- Students should read Chapter 13 from your text: Image Development: The Mystery and the Magic of Art, written by Sharon McCoubrey.
- Please make sketch notes for the chapter and try using at least two media for your page, starting with a background.
- Complete one additional personal drawing, trying one or more of the image development strategies from page 119. The final sketch journal will not be collected until November 22nd, but you should be developing a regular practice to show steady progress.
- You are also encouraged to start thinking about ideas for your major lesson plan assignment, due October 25th (three weeks from now). Our next few classes will help you create a lesson that you will be able to use in your own teaching practicum. Link here for a post with resource suggestions to get us started with LESSON PLANNING.
Anyone can be creative. Surround yourself with helpful influences, "play nice and work hard." (Austin Kleon)
What was the artist and writer Austin Kleon suggesting with his book title STEAL LIKE AN ARTIST ?


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In response to the opinion letter in the Aug. 5 edition of SJ-R submitted by Michael J. Hart, pastor of Peoples Church in Springfield, I suggest and invite you to come join the pickleball enthusiasts who frequent our city's public courts.
You mentioned that you are a former tennis player (many of us are); therefore, I believe that you too would fall in love with this new sport.
I found The Springfield Pickleball Club to be a very friendly, welcoming group of people. Players regularly travel from Jacksonville and Lincoln to join the fun at Iles and Douglas parks.
As a Springfield resident and taxpayer, I'm happy that our city parks are being utilized on a daily basis by people simply enjoying a healthy pastime.
Linda Curtis


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Exhibition and Shows
8 Feb 2019 to 24 Mar 2019
A stunning exhibition by Indigenous artists portraying their Country in the assorted colours of memory and season. The Artists have pooled their collective output to present a rich and diverse view of their Homelands. The colours represent landscapes from the coast to the desert, from the north to the south.
Art & Culture Shop
Free Entry
Event Times
8 Feb 2019 to 24 Mar 2019
Once Only Event
Japingka Gallery
47 High Street, Fremantle, 6160


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Despite news reports of great cops, it's getting harder and harder to respect law enforcement as we find out how many of them seem like psychotic monsters with badges and guns. Case in point, a cop in San Jose, CA, who took to Twitter to threaten protesters.
Buzzfeed uncovered some tweets by Officer Phillip White, who was complaining specifically about two mottos that have become prevalent in the protests about Ferguson, Missouri and Eric Garner in New York. The first is #BlackLivesMatter and the second is "I can't breathe" in reference to Garner's killing.
In the first tweet, White threatened, "Threaten me or my family and I will use my God given and law appointed right and duty to kill you." He ended the tweet with #CopsLivesMatter.
His next tweet was arguably even worse, when he said, "By the way if anyone feels they can't breathe or their lives matter I'll be at the movies tonight, off duty, carrying my gun."
Perhaps the most frightening thing is that this officer works with children and young adults. He addresses schools in San Jose on gang prevention and he's an assistant coach for a college basketball team.
In Berkeley, the women's basketball team wore shirts in support of the #BlackLivesMatter movement which named black men who have been killed by police.
Ironically, White tweeted, "This is BS. Free education. Don't forget who is paying for your school @CalWBBall me & other CA tax paying cops." I wonder who he thinks pays him?
The tweets have been deleted from White's account but he is still on the streets, armed, dangerous and with a giant chip on his shoulder. According to Buzzfeed:
"We have forwarded the matter up the chain of command for review," police spokesperson Sgt. Heather Randol told BuzzFeed News after being alerted to the tweets. "We can provide a response upon the completion of the review process."
You can register your complaints with the San Jose Police Department at 408-277-8900 or you can send them an email through their website. Please be more respectful than White.


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Article content
SAMARA, Russia — Yerry Mina scored on a header in the 74th minute and Colombia defeated Senegal 1-0 Thursday to advance to the World Cup knockout stage. Senegal became the first team ever eliminated by a new tiebreaker: number of yellow cards.
Poland defeated Japan 1-0 in the other group match. Japan and Senegal were tied on all tiebreakers — points (four), goal differential (zero) and goals scored (four) — except "fair play points," based on yellow and red cards. Japan had four yellow cards to Senegal's six.
tap here to see other videos from our team.
World Cup: Colombia wins, Senegal out on yellow cards Back to video
Colombia, which played in the quarterfinals four years ago in Brazil, finished atop its group and is the fourth South American team to advance. Senegal's elimination means no African teams are left.
Colombia and Japan go on to play opponents from Group G — either England or Belgium, who play at 2 p.m. ET Thursday.


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This concert has already taken place!
Haus im Park Theater Haus im Park
Ferne Klänge / Far-Off Sounds
Concert for People Suffering from Dementia
€ 5.00
It's often sounds and melodies more than anything that clear the fog of the subconscious and bring back memories we thought we had lost. Ensemble Resonanz plays for people suffering from dementia, and for their family and carers.
Please note: Tickets for this event are also available from the venue's box office (and occasionally when tickets are not available online). It is not possible to book wheelchair spaces online. Please contact us by phone at +49 40 357 666 66 or in person at the Elbphilharmonie ticket shops.
Ensemble Resonanz
Werke von Telemann, Dvořák, Copland u.a.
Blog & Streams
Promoter: HamburgMusik / Ensemble Resonanz / Körber-Stiftung
Haus im Park
Theater Haus im Park
Gräpelweg 8 / 21029 Hamburg
Getting Here
BegegnungsCentrum Haus im Park
Gräpelweg 8
21029 Hamburg-Bergedorf
Overground train S21: Bergedorf, 15 minutes by foot to venue
Bus 235 runs from Bergedorf station to bus stop »Rathaus«, 3 minutes by foot to venue
Opening Hours
Ticket office (»Theaterkasse«):
Tue-Fri, 14:00-18:00
Free parking is located in the side streets surrounding the theatre.
Some parking spaces for handicapped persons can be found in front of the main entrance of the theatre.
The ticket and box offices, toilets, catering and the building itself are accessible for persons with mobility disabilities. Designated toilets for disabled persons are also available.


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LA Traffic Mayhem control the road in LA
In this game you can control car traffic in LA city. First you need enter nick name and choose any city district in LA. If you to play in the first time, you can control only traffic at the Beverly Hills crossroad. Follow on ...start play!
Train Traffic Control logical strategy online game
You can be an Station Master. Manage train traffic. Don't let an accident with train crash. You must to prevent two trains on the same rail track. Stop the Train on station to earn some money. First station it's New...start play!
Game Rating: 5.00/ 5 (100.00%) 1 vote
Need something special? Search on the site 1ONLINEPLAY.com:


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Among the barrage of tables and statistics in a recent survey from the Law Society – Trends in the Solicitors' Profession, Annual Statistical Report 1995 – there is one sentence that stands out.
"There has been little change in the structure of private practice. Growth in the number of firms was almost entirely accounted for by an increase in sole practitioner firms," it says in the summary.
The survey points out that since 1985, the number of sole practitioner firms has grown by 34 per cent. The growth in technology is undoubtedly one of the factors behind this.
Nicholls & Co is a sole practitioner firm recently established in Norwich by Simon Nicholls. The practice, based at his home, is heavily reliant on a £4,000 hi-tech system.
Nicholls was previously a partner in a substanial provincial firm, which gave him plenty of office backup and support. The downside of this comfort was the substantial overheads. Nicholls' criminal and licensing work means that most of his time is spent outside the office, in court or visiting clients, with lots of time spent travelling between appointments, a particular problem in East Anglia.
As a result of his reliance on support from the office, Nicholls had not been closely involved with client accounting procedures, especially as most of his work was legal aid funded.
At first, he was concerned about starting up on his own. "The thought of going it alone was a daunting prospect," he says. "But not having staff costs and office leases to support has created a sense of freedom, and by making sensible use of modern office automation equipment I am always available to clients. I can now make much more efficient use of my time than ever before."
Nicholls has accumulated an impressive array of technology in his new office, including a mobile phone, a pager, a digital answering machine, a powerful desktop PC, a 2Mb Psion Series 3a portable computer, a Canon BJ10 EX portable printer, a modem and a Hewlett Packard Office Jet LX printer. The Hewlett Packard also serves as a copier, fax and page scanner.
"When surrounding yourself with technology you have to be careful that you use it wisely," says Nicholls. "I learned a lesson when I first started. I had a couple of answering services, one on my mobile phone and another on my machine in the office. It got a bit confusing and some people left more than one message for me on both machines. Now I've simplified things, and reduced my costs, by having only one message service on my digital answering machine. It advises the pager when a new message is received and also tells me the call length and cost.
"It is quite possible to run an entire criminal legal aid practice from the Psion," says Nicholls. He uses the Advocate system designed for the Psion by Julian Gibbons, a lawyer based nearby in Great Yarmouth.
"Using Advocate allows me to open files, complete attendance notes and value work at both standard and enhanced rates, no matter where I happen to be," says Nicholls. "There are programs for court diaries, mode of trial guidelines for all types of offences, time limits, PACE alarms, and all aspects of legal aid work including green forms, court duty solicitor, police station attendance, and magistrate and crown court appearances."
The Psion can be linked to the PC back at base using the PsiWin communications system. Nicholls has installed a Pentium PC running Windows 95 and Microsoft Office v7.
"I plan to establish electronic communications with a number of other sole practitioners, which will enable us to pass work to each other," says Nicholls. "I now have an email address and receive a lot of my instructions via fax.
"In fact, I prefer to send my letters by fax if possible. It's cheaper than the post, certainly less time consuming than addressing an envelope and it's 'greener' by saving paper."
Wherever possible, Nicholls transmits fully formatted word processing files to his associates, saving them time and money when it comes to redrafting documents.
"I look forward to using the telephone service on our local cable company when it becomes available later this year," he says. "I foresee many advantages from this service, including free local calls and quicker data transmission speeds."
The practice is a working example of the benefits technology can bring to modern practice. By carefully combining several technologies, all of which are available from high street shops, the sole practitioner can achieve the same levels of automation and efficiency enjoyed by many fee earners using large scale systems in bigger firms, at a fraction of the cost.
"I've not discovered a downside as yet," says Nicholls. "I had to progress from nothing to becoming Mr Technology in the space of a week. You have to prevent the technology becoming a barrier and make sure it is seen as a benefit. In fact, I am now more available to the public and my clients than ever before."
If the trend towards sole practitioners continues, the profession can expect to see many more products and services becoming available for this buoyant sector of the legal industry.
Nicholls & Co email address is email@example.com.
Advocate is available from Paradigm Software (01424) 212021 and costs £179 ex VAT. Annual charges are £60.


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FlexChecks Celebrates Earth Day 2019!
Happy Earth Day from the FlexChecks Team! To celebrate our wonderful earth, we took a few minutes out of our day to plant a Cherry Tree in the front of our office!
Sustainability is imortant to our team! From recycling to carpooling, we believe we should live and work in a way that will reduce our impact on our environment. That's why we chose to celebrate Earth Day 2019.
A few words on the Importance and Value of Trees:
Trees contribute to their environment by providing oxygen, improving air quality, conserving water, preserving soil, and supporting wildlife. According to the U.S. Department of Agriculture, "One acre of forest absorbs six tons of carbon dioxide and puts out four tons of oxygen. This is enough to meet the annual needs of 18 people."
Trees absorb and store rainwater which reduce runoff and sediment deposit after storms. This helps the ground water supply recharge, prevents the transport of chemicals into streams, and prevents flooding.
It's important for our payroll team to offer green alternatives to printing your reports and checks. We offer direct deposit, pay cards, and online reports. If you'd like more information on how easy it is to go paperless please contact us at 616-791-7900 or email us at email@example.com for more information!


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The Non-profit organization from Savannah, Georgia helps youth with opportunities in school and athletics.
Guayaquil, Ecuador, August 25 – 29— Youth Team USA comprised of 3 members of Performance Initative's Coastal Empire Weightlifting Club selected back in July to represent the United States at the Youth Pan American Games took home: Gold, Silver and Bronze and one American National record!
Kaiya Bryant, 3-time Youth National Champion and member of 2019 Youth World Team USA, in 49kg weight class, competed on Sunday. She snatched 66kg for a silver and clean/jerked 88kg earning her a gold in CJ, Gold overall and a new american record in clean & jerk. Kaiya,14, attends STEM Academy at Bartlett in Savannah.
Kye Bryant, brother of Kaiya and a 2018 Youth National Champion, a 11th grade student at Jenkins High School, division (55 kg) snatched 93 kg (bronze medal), clean and jerk 113 kg (silver medal) for a personal record total of 206 kg winning overall bronze.
"It's amazing to see our next generation of youth like Kaiya, Kye, and Nia excelling on the heels of sending several of our youth to their first year of college," exclaimed their coach, Kerri Goodrich
A veteran of the Youth Pan America Games, Nia Walker in the 81+kg division lifted her way to silver overall, silver snatch of 88kg, and silver clean/jerk with 117kg. Walker set personal records on her overall total of 205kg and both her lifts individually. Nia was also a member of the 2018 USA Youth Pan Am Team. Nia attends Johnson High School in Savannah currently in the 10th grade.
Goodrich, stated "As the school year starts these three athletes will have a lot to be proud of and we're excited to have our up-and-comping champions aspiring to be like them. Our programs inspire youth from elementary school age through college. We help with free tutoring, a Kids Cafe, providing dinner and a facility that helps create healthier hearts, minds and bodies."
The Performance Initiatives, a not for profit facility is supported by donations from individuals and businesses in our community. Please consider helping us support programs that help youth with opportunities in school, work and athletics.
Performance Initiatives (PI), Inc, strengthens hearts, minds and bodies of over 140 children annually through it's after school programs and partners with Second Harvest providing dinner at the Kid's Cafe. PI is home to Coastal Empire Weightlifting Club (CEWC) and Savannah CrossFit and provides classes for both adults and children. CEWC is a United States Amateur Weightlifting (USAW) Athlete Training Center.


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Too many loose-weight-tips around us, both scientifically and unscientifically.
Let see what we can do to reduce our body weight.
1. Build a Better Diet.
All meals are important, but breakfast is what helps you start your day on the right track. The best, heartiest breakfasts are ones that will fill you up, keep you satisfied, and stave off cravings later in the day. Aim to eat anywhere between 400 and 500 calories for your morning meal, and make sure you're including a source of lean protein plus filling fat (e.g., eggs, beans, unsweetened Greek yogurt, nuts, or nut butters) and fiber (veggies, fruit, or 100% whole grains). Starting your day with a blood sugar-stabilizing blend of nutrients will help you slim down without sacrifice.
Make sure that everything you're eating is whole — as in nothing processed or packaged. Since salt is a preservative, these are the foods that are highest in sodium — something to keep in mind when planning your meals. Plan on making sure that all items you choose are fresh. That means filling up on fresh fruits and veggies, whole grains, low-fat dairy, and lean protein.
Start your day with a cup of coffee or tea. Caffeine is a natural diuretic and an excellent source of antioxidants, which protect your cells from damage. While tea is also a natural diuretic, and types of herbal tea such as dandelion or fennel root can also lend a hand. And skip all sugary beverages. If you consume each of those beverages during the day, you'll have taken in at least 800 extra calories by nighttime — and you'll still be hungry.
Skipping meals will not make you lose weight faster. If a hectic day makes a sit-down meal impossible, stash an energy bar or a piece of fruit in your car or tote, keep snacks in your office desk drawer, and make a point of getting up to grab a nosh — anything that will keep you from going hungry! Going long periods of time without food does double-duty harm on our healthy eating efforts by both slowing down your metabolism, and priming you for another binge later in the day.
Munch on mineral-rich foods: potassium, magnesium, and calcium can help to serve as a counter-balance for sodium. Foods that are rich in potassium include leafy greens, most "orange" foods (oranges, sweet potatoes, carrots, melon) bananas, tomatoes, and cruciferous veggies — especially cauliflower. Low-fat dairy, plus nuts, and seeds can also help give you a bloat-busting boost. They've also been linked to a whole host of additional health benefits, such as lowering blood pressure, controlling blood sugar, and reducing risk of chronic disease overall.
Lastly, drink more water. Plenty of water to help expedite the process of ridding your body of excess sodium, you can (and should!) also consume high-water content foods. Reach for cucumbers, tomatoes, watermelon, asparagus, grapes, celery, artichokes, pineapple, and cranberries — all of which contain diuretic properties that will also help you stay full due to their higher fiber content.
2. Build Lifestyle.
The more lean muscle you have, the faster you'll slim down. How do you start strength training? Try some push-ups or a few squats or lunges. Use your free weights to perform simple bicep curls or triceps pulls right in your home or office. Do these exercises three to four times per week , and you'll soon see a rapid improvement in your physique.
Exercising at any time is good for you. But evening activity, may be particularly beneficial because many people's metabolism slows down toward the end of the day. 30 minutes of aerobic activity before dinner increases your metabolic rate and may keep it elevated for another two or three hours, even after you've stopped moving.


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1810 S Young Cir, Hollywood
(954) 922-1103
Offering the most unique dishes with daily fresh fish you will find this side of Tokyo. Try any one of our sake drinks with over 20 to choose from. The Sushi Room, a family owned sushi and sake lounge, has been open since 2006. Along with an excellen...
9409 Sheridan Street, Hollywood
(954) 885-8988
This restaurant certainly needs some marketing help! What's Asia 1? Come on can't they come up with a more appetizing and enticing name? Well, if you can get over the name of this restaurant then you will enjoy one of the best Northern Chinese foo...
3327 Sheridan St, Hollywood
(954) 987-1388
I go here often and the sushi is consistent. The owner/staff are always welcoming and they get very busy at night which can be a wait for the food, but that is because they don't make it ahead of time. If you go in the evening (after 7pm) be prepar...
1318 N Ocean Dr, Hollywood
(954) 929-0550
1935 Harrison St, Hollywood
(954) 921-1200
1924 Hollywood Blvd, Hollywood
(954) 921-1988
4401 Sheridan St, Hollywood
(954) 989-1515
3755 Hollywood Blvd, Hollywood
(954) 987-8569


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Mayor Dawn Zimmer Dares To Speak Truth To Power!!!
This is the time for all the residents of Hoboken…and of our whole state to find the courage to point out that the would be emperor is naked.
It is outrageous that one town gets 15 million to fix one boardwalk….but Hoboken only gets 300k to deal with the aftermath of Sandy. Something is seriously wrong with that picture….and now we know for certain what many of us have suspected all along.
What Mayor Zimmer is doing takes what JFK wrote about in his book, "Profiles in Courage".
There is no certainty as to the outcome of all of this. If the forces of darkness and corruption prevail….there will be retribution for decades to all who have stood up against them.
This is now about much more than simply bridgegate, or about Hoboken or even just about governor Christie………it's about the way we do politics in New Jersey.
The politics of retribution and distribution are toxic and wasteful. The golden goose is starving to death….we never could afford the corruption.
Let's use this systemic mess as an opportunity to form a statewide movement to radically transform NJ politics in the direction of justice and and common human decency.
This is a time for all people of good will who care about truth, justice and good government to stand with Mayor Zimmer in speaking truth to power.
The people, united…can never be defeated.


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Let's face it, a nice creamy chocolate cake does a lot for a lot of people; for you too!" Love and Cheesecake is a well-known shop for cakes in Mumbai. They add flavors and designs to your wedding through their unique and beautiful cakes which are always loved by every single couple as well as by all... See +
With our vast amount of experience and in-depth interaction with various clients, we have become the vendor of choice and a one stop solution for customized cakes, chocolates, theme parties, events and packaged snacks solution. Welcome to the world of chocolates, cakes and fantasies where your experience... See +
Eat Drink Design is a boutique catering service, where design and food intertwine with the promise to take you on an unforgettable culinary experience. EDD offers personalised global-cuisine menus, singular presentation styles and ambient designs for all events. with a team of highly skilled Chefs,... See +
Good Food Concept is one of the pioneer caterers in Mumbai providing bespoke catering services since 20 years. We at GFC caterers believe in delivering wonderful food and a first class catering service for all occasions. We always strives to make every event a special one which makes us as one of the... See +
It all started in the late fifties when we made our debut in the world of catering. Since then, we have been spicing up the culinary world with detectable vegetarian food. We started off initially by serving our clients Indian – vegetarian delights in the most authentic preparation thus winning their... See +
Crumbs to Gourmet offers our customers a Private Dining experience in their own homes or choice of venue. Joel is an accomplished chef who will create a bespoke menu for your gastronomic pleasure. He will provide you with fine food and a discreet and elegant service, leaving you free to relax and enjoy... See +
We are one of the most prominent enterprises engaged in providing professional Catering Services to the clients. Our services are appreciated for hygiene, authentic cuisines preparation and individualized & prompt delivery. the professional catering services offered by us fulfill the needs of the... See +
We take care of all the details, from preparation to clean up. Our team of highly qualified professionals will make any event a pleasure. From weddings to cocktail parties, or anything in between, Dove Entertainment is your best choice. Our food reflects our commitment to exceptional quality. Our menu... See +
D Cake Creations offer designer cakes for every celebration in your life – Engagement, Wedding, Anniversary, kid's birthdays, milestone birthdays, baby shower or even corporate parties – all personalized to suit your requirements or theme. Rinku ensure that each cake is made to the highest of standards,... See +
With recipes that have been passed on for generations, Not Just Desserts offers a wide range of exquisitely crafted desserts, perfect for all types of celebrations. Whether an occasion or 'just because', their desserts are sure to spread happiness to you and your loved ones. Each dessert offers the... See +
Foodlink Banquets & Catering Pvt. Ltd. was established in 1998 with a vision to be India's first integrated premium event catering company. It has now become the first choice of the most discerning clients ranging from the country's blue blooded to the star studded. Over the last 15 years, Foodlink... See +
Mini Punjab's original owners were pioneers who began the "Koliwada Fish Fry" taste revolution and singlehandedly made it a national and international favourite. Today, while there are many who serve "Koliwada Jhinga Fry" and "Koliwada Rawas Fry", those of discerning taste know that there is only... See +
A Culinary Journey Founded On Passion, Driven By Perfection Being in the hospitality business for a long time, has given us an unparalleled knowledge of the Indian Market, Culture And Conditions. We have a proud history of delivering truly exquisite cuisine accompanied by innovative and dedicated... See +
After declining the offer to deliver cakes to about 30 odd people, they thought they could design and delivery even better designer cakes in and around Mumbai, they got a cake designer on-board, yes in an IT startup, they have cake, cupcake, pastry chef on-board. They design themed cakes and cupcakes,... See +
'The Gaurav Caterers", We are very pleased to introduce ourselves as one of the leading professional caterers in Mumbai, Maharastra, since 1992. initially commenced by 3 brothers Mr. Shankar, Mr. Satish & Mr. Sushil Harwani, to manage Food and Beverage Operation in a well known Landmark of Mumbai... See +
Known for his conceptualization more than anything else. The first person to start the trend of pool parlours in Mumbai with his brand Crazy Balls & also giving the Film Industry Fantasy, the brand associated with luxurious make-up vans and house-on-wheels, or internationally known as RVs. He has... See +
After the tremendous success of his signature properties "Amadeus", "Cafe At The NCPA" and "Umame" in South Mumbai, Farrokh Khambata's journey comes full circle as he opens a new avatar of "Joss", his signature Modern Oriental restaurant at Santacruz, Mumbai. Ten years ago Chef Farrokh Khambata introduced... See +
Welcome to the Li'l White Oven, your one stop blog for all baking recipes, resources, tips and tricks, and also a place where you can order customized cakes for your loved ones. Li'l White Oven is not just a business name. Hardly. It is the story of how one tiny little white colored oven changed... See +
Hangout Cakes and More believes in "Everything sweet! And even more!" Hangout Cakes and More is available everywhere in Mumbai, they serve you the way you want for your dream day. They have an experience to full fill the cake requirements for weddings, engagements, birthdays, parties and all. They prepare... See +
Moments of celebration and special occasions - Celebrate it with LE KITCHEN! At LE KITCHEN, we offer our customers options of choosing from a simple pre-packaged catering menu or a customized menu that you could create from our elaborate list of menu suggestions. Of course, highest quality food is... See +
Neeta Gurbaxani is her name. A passionate cake maker who extends her sweet personality to everything she bakes, it's no wonder that she's simply known as The Cake Lady! A graduate of the Hotel Management Institute, she began her career working at the Taj Patisserie, Sea-Rock Hotel and trained with... See +
At Vegan Bites our aim is to make it easy and convenient for you to make healthy meal choices whether you are at home or at work. Vegan Bites is dedicated to serving you Whole, Fresh Plant-Based Meals. Our team of chefs, nutrition experts and doctors work together to ensure that your meal is not just... See +
Trupti Group, a name synonymous with expertise, excellence and unparalleled quality has seen a tremendous growth over the last three decades. What started out as a vision of two brothers, Kanubhai Suchak and Sureshbhai Suchak in 1980 is now a legacy within the wedding and hospitality sector. The... See +
We provide a comprehensive outdoor catering service including a range of foods and drinks coupled with our outstanding service and trained staff. We can arrange cocktail parties for any number of guests and any occasion; Weddings, Birthdays, Office parties, Exhibitions, etc. We make every effort to... See +