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To study the regulatory and functional differentiation between the mesophyll (M) and bundle sheath (BS) cells of maize (Zea mays), we isolated large quantities of highly homogeneous M and BS cells from newly matured second leaves for transcriptome profiling by RNA sequencing. A total of 52,421 annotated genes with at least one read were found in the two transcriptomes. Defining a gene with more than one read per kilobase per million mapped reads as expressed, we identified 18,482 expressed genes; 14,972 were expressed in M cells, including 53 M-enriched transcription factor (TF) genes, whereas 17,269 were expressed in BS cells, including 214 BS-enriched TF genes. Interestingly, many TF gene families show a conspicuous BS preference in expression. Pathway analyses reveal differentiation between the two cell types in various functional categories, with the M cells playing more important roles in light reaction, protein synthesis and folding, tetrapyrrole synthesis, and RNA binding, while the BS cells specialize in transport, signaling, protein degradation and posttranslational modification, major carbon, hydrogen, and oxygen metabolism, cell division and organization, and development. Genes coding for several transporters involved in the shuttle of C4 metabolites and BS cell wall development have been identified, to our knowledge, for the first time. This comprehensive data set will be useful for studying M/BS differentiation in regulation and function.
C4 plants, with few exceptions, require the coordination of the mesophyll (M) and bundle sheath (BS) cells, arranged in a wreath structure called Kranz leaf anatomy (Hatch and Agostino, 1992), to confer high rates of photosynthesis. The initial carboxylation phase of the C4 pathway takes place in the M cells, while the decarboxylation phase is restricted to the BS cells. The high photosynthetic capacity of C4 plants implies a massive efflux of C4-related metabolites between M and BS cells and between the cytosol and organelles in each cell type (Weber and von Caemmerer, 2010).
Although research in the past few decades has greatly increased our understanding of the biochemical reactions and the enzymes involved in the C4 pathway of photosynthesis, little is known about the specific genes involved in the development of the Kranz leaf anatomy, the C4 biochemical pathway, and the underlying regulatory mechanisms for the high-level expression of C4-specific genes in a cell-, organ-, or development-specific manner. With the advancement in genomics, the genomic sequences of several C3 and C4 model plants have become available. These advances have allowed in-depth comparative proteomic and transcriptomic analyses of the whole leaves of typical C3 and C4 plants and their closely related C3-C4 intermediate species of Cleome and Flaveria (Gowik et al., 2011). These comparative studies allow deduction on how many genes are required to make a C4 plant and possibly on how they may have been regulated at the genetic level. In addition, attempts have been made recently to characterize the transcriptomic profile of maize (Zea mays) leaf in a development-dependent manner (Li et al., 2010). Also, the laser-capture microdissection (LCM) technique was used to isolate both M and BS cells from mature maize leaves for transcriptome analysis. However, LCM has a limitation in the amount of cells that can be isolated and has the potential of cross-contamination.
To gain a comprehensive understanding of the C4 photosynthesis pathway and related processes, we have modified current methods to isolate large quantities of highly purified M and BS cells from mature maize leaves for RNA extraction and detailed transcriptome analyses of M and BS cells. Our study reveals the functional differentiation and coordination of the two cell types in various metabolic pathways and provides insights into the possible regulatory network underlying the development of Kranz leaf anatomy and the operation of the photosynthetic and other metabolic pathways in the NADP-malic enzyme (ME) subtype C4 plants.
Isolation of Highly Pure M Protoplasts and BS Strands
In this study, highly purified mesophyll protoplasts (MP) and bundle sheath strands (BSS) were obtained in large quantities from newly matured second leaves (Supplemental Fig. S1) for transcriptome analyses. Examination by light microscopy showed that the mechanically purified BSS was attached with very little M tissue, except M cell wall and vascular and epidermal tissues. To further assess the purity of the isolated cells, the activities of two key C4 photosynthesis marker enzymes for M and BS cells were assayed. On a chlorophyll basis, the high activity of NADP-ME, a key C4 acid decarboxylase located in the BS cells of maize, was almost exclusively associated with the BSS isolated (Supplemental Table S1). On the other hand, the high activity of phosphoenolpyruvate carboxylase (PEPC) was mainly associated with the MP isolated by enzymatic digestion, although a low activity of PEPC was also detected in the BSS isolated, which might be due to the presence of the C3-type PEPC in the BS cells. Immunoblot analysis using specific antibodies raised against the maize proteins also demonstrated the high homogeneity of the two cell types isolated (Supplemental Fig. S2).
Raw Read Processing and Definition of Expressed Genes
To clean the sequencing raw reads, we deleted reads that contain any N (unknown nucleotide) or have a poor quality score (Table I). Using Tophat version 1.3.3 (Trapnell et al., 2009; http://tophat.cbcb.umd.edu/), we found 78% and 79% of the cleaned reads can be mapped to the maize B73 genome (Maize Genome AGP version 2, release 5a.59).
We used Cufflinks version 1.2.1 (Trapnell et al., 2010; http://cufflinks.cbcb.umd.edu/) to calculate the expression level of each gene in terms of reads per kilobase per million mapped reads (RPKM; Mortazavi et al., 2008). RPKM is normalized by the length of the exon and the total mappable reads and can be used to quantify the expression level of a gene from the mapped read count. A total of 52,421 annotated genes with at least one read were found in the two transcriptomes (Table II). (All genes with at least one read are listed in Supplemental Data S1 with their RPKM values.) The maximum RPKM were 35,421 and 31,561, and the median RPKM were 0.55 and 0.73 in the M and BS cell transcriptomes, respectively. Only approximately 2% of the genes had a very high expression level (RPKM ≥ 100) in either M or BS cells.
The quality of the transcriptomes, the potential contamination of BS cells by epidermal and vascular bundle tissues, and the potential effects of long incubation time during the isolation of M protoplasts are discussed in Supplemental Materials and Methods S1 and Supplemental Fig. S3.
Following the criterion of Li et al. (2010), we defined an "expressed gene" as a gene with RPKM > 1 in the transcriptome. In all analyses below, only expressed genes were considered. If the Filtered Gene Set (FGS) was chosen, we identified 14,972 and 17,269 expressed genes in the M and BS transcriptomes of maize, respectively (Fig. 1A). In total, we identified 18,482 genes expressed in either the M or BS transcriptome or both, which is 1,844 genes more than were identified by Li et al. (2010; Fig. 1A). If the Working Gene Set (WGS) was chosen, then we gained an additional 2,224 expressed genes (Fig. 1A).
Low Degrees of Cross-Contamination between the Two RNA Samples
It is important to know the degree of cross-contamination between the two transcriptomes, because cross-contamination can affect the estimation of the number of expressed genes in a transcriptome (one cell type) and the number of differentially expressed genes between the two transcriptomes (two cell types). To address this issue, we used the expression levels of 10 selected C4 marker genes to estimate the degrees of cross-contamination in the two RNA samples from the isolated M cells and BS cells (Table III). The proteins encoded by these genes play key roles in C4 photosynthesis and are assumed to be exclusively or almost exclusively expressed in only one cell type (Supplemental Data S1). Among the 10 selected genes, NADP-ME (GRMZM2G085019), DCT2 (for dicarboxylate transporter; GRMZM2G086258), PCK (GRMZM2G001696), and two rbcSs (GRMZM2G098520 and GRMZM2G113033) are supposed to be BS specific, while PEPC (GRMZM2G083841), CA (GRMZM2G121878), NADP-MDH (GRMZM2G129513), OMT (for 2-oxoglutarate/malate transporter; GRMZM2G383088), and PPDK-RP (GRMZM2G131286) are supposed to be M specific. Using Equations 2 and 4 as shown in "Materials and Methods," we obtained an upper estimate of c1 = 1.2% for the contamination rate of M cells by BS cells and an upper estimate of c2 = 4.5% for the contamination rate of BS cells by M cells (Table III). These are considered the upper estimates because the assumption of exclusive expression of a key C4 gene in a cell-specific manner (e.g. PEPC in M cells) may not be absolutely true. In any case, these calculations suggest that the degrees of cross-contamination in our two RNA samples were reasonably low, which is crucial for us to accurately determine the expression profile of a given gene in a cell-specific manner.
We checked the above conclusion on the purity of our isolated M and BS cells by quantitative reverse transcription-PCR (Supplemental Table S2 and S3). For the three BS-specific genes studied (rbcS-2, rbcS-4, and NADP-ME), the respective M/BS expression ratios were 0.001, 0.049, and 0.004, which are indeed similar to the ratios obtained from the transcriptome data (0.022, 0.022, and 0.007). These data support the conclusion of very low contamination of our M cell sample by BS cells. For the three M-specific genes studied (PEPC, NADP-MDH, and OMT), the respective BS/M ratios were 0.055, 0.050, and 0.037, which are also very close to the BS/M ratios of 0.048, 0.039, and 0.045 from our transcriptome data (Supplemental Table S2), supporting an estimate of approximately 4.5% contamination of our BS sample by M cells.
Li et al. (2010) had reported two sets of deep-sequencing transcriptomic data from the maize M and BS cells isolated by LCM. In their data, nine out of the above 10 C4 marker genes, except PPDK-RP (GRMZM2G131286), were listed as cell type-enriched genes. Applying the same equations to analyze their data, we estimated the upper contamination rates of c1 = 9.2% and c2 = 21.6% for their M and BS cell preparations. These results indicate that our isolation methods caused a much lower level of cross-contamination than the LCM method they used. Consequently, our data would give a more accurate estimation of genes differentially expressed between the M and BS cells.
Genes Differentially Expressed between the Two Cell Types
To estimate the differential expression of genes between the two cell types, we defined the degree of cell specificity of gene i as Ri = |mi – bi|/max(mi, bi), where mi and bi represent the RPKM of gene i in the M and BS RNA samples, respectively. By this definition, Ri = 1 if gene i is exclusively expressed in only one cell type, and Ri = 0 if gene i is equally expressed in the two cell types. To define "differentially expressed genes," we used the criterion of Ri = 0.8, which corresponds to a 5-fold difference in RPKM value between the two cell types. This criterion is more stringent than the criterion used by Li et al. (2010); 72% of their differentially expressed genes had only 4-fold or smaller differences. In the differentially expressed gene set, there were 1,545 and 3,422 genes enriched in the M and BS transcriptomes, respectively (Fig. 1B). That is, 4,697 (1,545 + 3,422) genes, or approximately 25% of all expressed genes in the FGS (18,482), were differentially expressed between the two cell types. In contrast, Li et al. (2010) identified only 3,427 (2,028 + 1,399) differentially expressed genes, or approximately 21% of all expressed genes (16,638), even though they used a looser criterion (Fig. 1B). The lower percentage could be due to the high cross-contamination rates in the study of Li et al. (2010; see above). In our data, there were many more BS-enriched genes than M-enriched genes (Table II). This observation is opposite to the Li et al. (2010) observation of more M- than BS-enriched genes. Only 30% and 16% of M- and BS-enriched genes in this study overlapped with the Li et al. (2010) data (Fig. 1B). Thus, many more cell-specific genes have been identified from our study. For genes with Ri > 0.99, which can be taken as truly cell-specific genes, there were only 65 genes in M cells but 688 genes in BS cells (Supplemental Data S2). This result suggests that maize BS cells have assumed a more important metabolic role in C4 photosynthesis and other metabolic processes.
The major difference in photosynthetic biochemistry between C3 and C4 plants is that in C4 plants the CO2-concentrating mechanism and the Calvin cycle are divided between M and BS cells. To achieve an effective CO2-concentrating mechanism, genes that are involved in carbon fixation, such as PEPC (GRMZM2G083841), CA (GRMZM2G121878), NADP-MDH (GRMZM2G129513), and PPDK-RP (GRMZM2G131286), and plastid membrane transporters such as OMT (GRMZM2G383088), are all enriched or specifically expressed only in the M cells. In our M transcriptome, the RPKM values for PEPC, CA, NADP-MDH, PPDK-RP, and OMT were 9,485, 6,692, 2,621, 476, and 988, respectively, much higher than the corresponding values 431, 281, 95, 22, and 74 in the BS transcriptome (Table III). Therefore, the cell-specificity Ri values for these five genes were 0.95, 0.96, 0.96, 0.95, and 0.93, respectively. (These Ri values were probably underestimated because of the potential contamination of our BS cell sample by M cells.) On the other hand, the genes involved in releasing CO2 from C4 acid decarboxylation, such as NADP-ME (GRMZM2G085019) and PCK (GRMZM2G001696), plastid membrane transporter, such as DCT2 (GRMZM2G086258), and the Calvin cycle, such as rbcSs (GRMZM2G098520 and GRMZM2G113033), were all enriched or specifically expressed in BS cells, each with Ri > 0.98 (Table III). Taken together, these results indicate that most of the known C4 genes are very tightly regulated and specifically expressed in M or BS cells in mature maize leaves, allowing an effective CO2-concentrating mechanism through effective carboxylation and decarboxylation in the two cell types. An exception to this rule is PPDK (GRMZM2G306345): it is preferentially expressed in M cells with a Ri of only 0.78. This is consistent with the biochemical study by Aoyagi and Nakamoto (1985), which found substantial activity and protein of PPDK present in the maize BS cells. The conversion of phosphoenolpyruvate (PEP) to pyruvate and ATP catalyzed by the reversible PPDK is another metabolic way for cells to produce ATP.
There is strong biochemical evidence that the PCK subtype C4 photosynthesis plays a substantial role in some NADP-ME subtype C4 plants, such as maize (Wingler et al., 1999; Leegood and Walker, 2003). Consistent with this notion, PCK was found to be expressed at an extremely high level and exclusively in BS cells (i.e. Ri = 1.00; Table III). Two other enzymes are also required to support the operation of the PCK decarboxylation system in both cell types, namely aspartate aminotransferase (AspAT) and alanine aminotransferase (AlaAT). There are two major paralogs of AspAT found in maize with a high level of expression: one (GRMZM5G836910) is M specific (Ri = 0.97), which encodes a chloroplast enzyme, confirming the biochemical results of Gutierrez et al. (1974), while the other (GRMZM2G094712) is BS enriched (Ri = 0.79), which encodes a mitochondrial enzyme (Supplemental Data S3). In contrast, the two AlaAT paralogous genes showed moderate expression levels, both with an M cell preference. The presence of a large amount of AspAT transcript in M cells is necessary for the operation of the PCK decarboxylation system in maize leaves, as OAA is very labile and once produced should be taken up quickly by the M chloroplast for reduction to malate by NADP-MDH or transaminated to Asp by AspAT.
Functional Differentiation between M and BS Cells and Identification of Genes Involved in Kranz Structure Formation
For the 1,545 M-enriched and 3,422 BS-enriched genes identified, their biological functions were further elucidated using MapMan (http://mapman.gabipd.org/): 71% of the enriched genes (3,358 genes: 1,015 M enriched and 2,343 BS enriched) have been assigned to known biological processes or metabolic pathways. Except for those that have been classified as "others" (14%), the three most abundant functional groups are protein metabolism (13%), RNA metabolism (11%), and transport (6%; Fig. 1C). The other abundant functional groups are miscellaneous (5%), signaling (4%), cell (3%), stress (3%), hormone metabolism (3%), photosynthesis (2%), and development (2%; Fig. 1C). When considering the functions of these genes in M or BS cells, we identified 15 cell type-enriched functional groups using the Fisher exact test (P < 0.05 for each group; Fig. 1D). The difference in the number of cell type-enriched genes between M and BS cells within each function is quite large, except for the protein-targeting category. Overall, the number of BS-enriched genes (3,422) is much higher than that of M-enriched genes (1,545). As expected, the transcripts of genes coding for photosynthesis light reaction components were more abundant in M than in BS cells. Interestingly, over 90% of the genes related to protein synthesis (for ribosomal proteins) and protein folding were preferentially expressed in M cells, while those involved in protein degradation were predominantly expressed in BS cells. These observations indicate that many proteins were synthesized and folded in M cells but eventually degraded in BS cells, which surround the vasculature. This arrangement facilitates the transport of amino acids released from protein degradation to other plant parts. Genes involved in RNA regulation of transcription, transport, signaling, cell division, organization and vesicle transport, development, and major carbon, hydrogen, and oxygen metabolism were predominantly expressed in BS cells, but genes involved in secondary metabolism, tetrapyrrole synthesis, and RNA binding showed an M cell expression preference. Most of the secondary metabolites accumulate in the epidermis cells, adjacent to M cells.
For the 85 BS-enriched genes involved in development, we found that 22 genes encode the nodulin MtN3 and MtN21 family proteins, which were originally identified as root nodule proteins of the legume Medicago truncatula (Gamas et al., 1996). MtN3 and MtN21 also have recently been shown to be crucial for exine pattern formation and cell integrity of microspores (Guan et al., 2008) and secondary wall formation in xylary and cotton fibers (Ranocha et al., 2010), respectively. These genes code for transmembrane proteins involved in the cell wall development of specialized plant cells that require stringent impermeability to specific substances (e.g. oxygen). Therefore, the counterparts in maize leaves might be involved in the development of the BS cell wall, which plays a key role in preventing the leakage of CO2 released from C4 acid decarboxylation in order to maintain a high CO2 concentration in BS cells (Hatch et al., 1995). At present, little is known about the biochemical composition of the C4 BS cell wall. For the first time, to our knowledge, our data suggest that C4 plants may have adopted the exine composition of microspore and pollen for its impermeability to a number of substances, such as CO2. Further analysis on the expression profile of these genes along the development of the Kranz anatomy in C4 leaves should provide insights into their functional roles.
Cell Type-Enriched Transcription Factors
Among the 2,056 annotated transcription factor (TF) genes in maize, 654 and 820 were expressed in M and BS cells, respectively (Table II). The median RPKM for all expressed TFs in the two cell types were 5.7 and 7.1, respectively (Supplemental Data S4). We identified 53 TF genes as M enriched and 214 TF genes as BS enriched (Table II), which, according to the TF family database PlantTFDB version 2 (Zhang et al., 2011), belong to 16 and 38 TF families, respectively. Six of the BS-enriched TFs were assigned to two different families simultaneously, because of two different types of DNA-binding domains in a protein. Three TFs, MYB59 (GRMZM2G130149), MYB111 (GRMZM2G305856), and another MYB family protein (GRMZM2G002128), belong to both MYB and MYB-related families. The other three TFs are AGL12 (GRMZM2G105387), which belongs to both M-type and MIKC families, RPL (GRMZM2G154641), which belongs to both HB-other and TALE families, and a zinc finger (B-box type) family protein (GRMZM2G075562), which belongs to both CO-like and DBB families. Interestingly, in 11 of the families with cell type-enriched TF genes, all members showed BS enrichment, whereas none has all members showing M enrichment (Fig. 1E). This observation suggests that many TF families have a cell type preference, indicating a differentiation in regulatory network for the functional differentiation of the two photosynthetic cell types in maize leaves. Comparison of the distributions of cell type-enriched TF families between this study and Li et al. (2010) shows some similarities but many differences. For example, Li et al. (2010) identified only two ARF genes enriched in M cells and none enriched in BS cells (Fig. 5d in Li et al., 2010), while we identified seven ARF genes enriched in BS cells but none enriched in M cells (Fig. 1E). Cross-contamination of isolated M and BS cells will lead to underestimation of cell-specific genes.
We searched the literature to identify the functions or pathways where a TF might be involved (Table IV). Most M-enriched TFs are involved in the pathways of responses to environmental stimuli. For example, the three paralogs of DREB1A (GRMZM2G380377, GRMZM2G069146, and GRMZM2G069126) are related to the function of cold acclimation (Novillo et al., 2007). The two ERF TFs, CBF4 (GRMZM2G124037) and ERF7 (GRMZM2G089995), are involved in the pathways of responses to drought and abscisic acid (Haake et al., 2002; Song et al., 2005), respectively. In addition, three TFs in the NAC family and two in the WRKY family are induced by wounding and pathogens (Collinge and Boller, 2001; Xu et al., 2006; Zheng et al., 2006), respectively. Two other M-enriched TFs were GLK1 (GRMZM2G026833), which is involved in pathways of chloroplast development specific to M cells (Nakamura et al., 2009), and ARR10 (GRMZM2G013612), which is related to protoxylem differentiation (Yokoyama et al., 2007; Table IV).
Very different from the M-enriched TFs, most BS-enriched TFs are related to development or the biosynthesis of cells and organelles. These functions include chlorophyll biosynthesis, secondary cell wall biosynthesis, cell division, cell differentiation, or development of chloroplast and leaves. The observations also match some functional preferences of BS-enriched genes: categories of development and cell (division, organization, vesicle transport, etc.; Fig. 1D). Three TFs, MYB20 (GRMZM2G055158), MYB52 (GRMZM2G455869), and MYB54 (GRMZM2G077147), in the MYB family, and ATAF1 (GRMZM2G179049) in the NAC family, are related to secondary cell wall biosynthesis (Zhong et al., 2006, 2008). Two TFs, TCP4 (AC205574.3_FG006) and TCP5 (GRMZM2G120151), in the TCP family are related to cell division (Cubas et al., 1999; Aggarwal et al., 2011). The GRAS TFs, RGA1 (GRMZM2G023872) and SHR (GRMZM2G132794), are related to asymmetric cell division (Truong et al., 1997; Heidstra et al., 2004). Two bHLH TFs, PIL5 (GRMZM2G165042) and FMA (GRMZM2G162450), are related to chlorophyll biosynthesis and cell differentiation (Huq et al., 2004; Gray, 2007), respectively. Three ARF TFs, ARF3 (GRMZM2G056120), ARF6 (GRMZM2G317900), and ARF8 (GRMZM2G078274), are related to leaf or floral organ development (Finet et al., 2010; Tabata et al., 2010; Table IV).
Interestingly, the ERF family was the only one that showed a larger number of M-enriched TFs than BS-enriched TFs. Many of these M-enriched ERF TFs have been shown to play a role in response to environment stimuli, but none of the BS-enriched ERF TFs are known to play such a role. Our data also showed that GOLDEN2 (GLK2; GRMZM2G087804) was preferentially expressed in BS cells of mature maize leaves at a moderate level (RPKM = 29) with Ri = 0.78 (Supplemental Data S1). GLK2 has been shown to be involved in maize chloroplast development in the BS cells independent of light, but it is not involved in rbcS accumulation (Hall et al., 1998; Cribb et al., 2001). On the other hand, GLK1 was highly and preferentially expressed in M cells (Ri = 0.89). These data suggest that GLK1 may regulate plastid development in M cells, while GLK2 may regulate plastid development in BS cells of C4 leaves.
More Genes Are Expressed in M and BS Cells Than Previously Known
Recently, Li et al. (2010) examined the transcriptomes of M and BS cells isolated from fully expanded third maize leaves by LCM. About 41 million 32-bp sequencing reads from the two cell transcriptomes were obtained. In this study, we generated approximately 370 million 120-bp reads from M and BS RNA samples (approximately 177 and approximately 193 million reads, respectively). Our data allowed the identification of 18,482 expressed genes in total, while Li et al. (2010) detected only 16,638 expressed genes. Another possible reason for the above difference between the two studies is that Li et al. (2010) used an earlier annotation version (APGv1) and only included genes with complementary DNA (cDNA) support, while we used APGv2. As a result, the total number of expressed genes obtained from our data is higher than that obtained by Li et al. (2010).
In addition to the expressed genes in the FGS, we detected an additional 2,224 expressed genes in the WGS (Fig. 1A), with the exclusion of transposable elements, gene fragments, and pseudogenes. Many genes in this additional gene set were highly expressed, with RPKM > 100 for 85 M-enriched genes and 86 BS-enriched genes. The highest RPKM values in this set were 4,900 (GRMZM5G809350; a paralog of LHCII type 1 CAB2) and 31,561 (GRMZM2G142891; a paralog of β-glucosidase aggregating factor) in M and BS cells, respectively. Furthermore, we detected 178 M-enriched and 610 BS-enriched genes in the WGS. Although there is no MapMan annotation for the genes in the WGS, 263 of the 2,224 expressed genes in the WGS are found to have one or more Gene Ontology terms (http://www.geneontology.org/); the five most abundant terms in molecular functions are ATP binding (23%), ice binding (13%), zinc ion binding (12%), DNA binding (10%), and protein binding (10%). Three M-enriched genes (GRMZM2G411457, GRMZM2G030616, and GRMZM2G152576) are related to light reaction in photosynthesis (Supplemental Data S1).
Notably, our data reveal more genes expressed in BS than in M cells in mature maize leaves. Expressed genes from vascular tissues (e.g. sieve element) in the BS cell preparation might have contributed to the category of BS-expressed genes, as the BSS isolated in this study contained the vascular tissue (Supplemental Table S4) and sieve elements are active cells for transport function. Thus, as discussed above, a small portion of the RNA from vascular tissues might have contributed to our BS transcriptome. However, it is most likely that more genes are expressed in BS cells than in M cells because of functional necessity, in agreement with the observation that the BS cells in C4 plants play much more important roles than the M cells in various physiological processes (Majeran et al., 2005; Leegood, 2008). In addition to genes related to C4 acid decarboxylation, expression of specific genes related to the metabolism of photorespiration, nitrate, sulfate, ammonium, starch, ion transport, secondary metabolites, and cell wall structure is largely confined to BS cells.
Light Reaction, Carbon Fixation Pathway, and Related Transporters
Many of the maize genes coding for the PSI components are highly expressed in both cell types, but with higher expression levels in M cells (Supplemental Data S3). In contrast, many of the maize genes coding for the PSII components are highly and predominantly expressed in M cells, with Ri > 0.9 for most of the genes. Transcripts for the two genes coding for ATP synthase, however, are found in both cell types. Most of the NDF (for NDH-dependent cyclic electron flow) genes coding for the iron-sulfur cluster-binding protein involved in cyclic electron transport exhibited a preferential expression in BS cells, with Ri ranging from 0.67 to 0.86, whereas two NDF genes (GRMZM2G865543 and GRMZM2G162233) showed a moderate preference in expression in BS cells, with Ri of 0.38 and 0.36, respectively. These data are in agreement with the observations that maize BS cells possess a higher capacity for cyclic electron transport (Ivanov et al., 2007).
Based on the expression profiles of C4-specific genes (Table III; Supplemental Data S1 and S3), we propose the partition of the key biochemical reactions of the C4 photosynthesis pathway between M and BS cells of maize, an NADP-ME subtype C4 plant (Fig. 2). Hydration of CO2 to bicarbonate, generation of PEP from pyruvate, carboxylation of bicarbonate to OAA, and its subsequent transamination to Asp or reduction to malate in the chloroplast all take place in the M cells. In contrast, decarboxylation of OAA to PEP and CO2 and decarboxylation of malate to pyruvate and CO2 occur in the cytosol and chloroplast of BS cells, respectively. The high expression levels of NADP-ME and PCK suggest that the PCK decarboxylation system plays a substantial role in maize C4 photosynthesis, consistent with previous biochemical studies (Furumoto et al., 1999, 2000; Wingler et al., 1999; Furbank, 2011). The dual decarboxylation system in some NADP-ME subtype C4 plants, such as maize, probably makes them the most efficient photosynthetic plants.
High rates of photosynthesis by C4 plants imply high fluxes of metabolites intercellularly and intracellularly. In C4 plants, most of the photosynthetic metabolites are known to be transported symplastically between M and BS cells through plasmodesmata. However, within each cell type, transporters on the chloroplast envelope must be active to substantiate the heavy traffic of related metabolites between cytosol and chloroplast. A maize gene coding for a 2-oxoglutarate/malate translocator (ZmpOMT1; GRMZM2G383088) was highly and predominantly expressed in M cells, and a dicarboxylate transporter gene (ZmpDCT1; GRMZM2G040933) was also preferentially expressed in M cells at a moderate level. Thus, these two transporters may be responsible for the uptake of OAA and the export of malate by M chloroplasts, respectively (Taniguchi et al., 2004). In contrast, the transcript for another maize gene coding for DCT (ZmpDCT2; GRMZM2G086258) was highly and almost exclusively expressed in BS cells, which may be responsible for the import of malate by BS chloroplasts, as suggested previously by Taniguchi et al. (2004).
In C3 plants the chloroplast phosphate translocator triose phosphate/phosphate translocator (TPT) is thought to have a central role in the partitioning of fixed carbon between starch in the chloroplast and Suc in the cytosol. Inorganic phosphate is taken up by the translocator in exchange for triose phosphate, which is then metabolized to Suc in the cytosol. In NADP-ME subtype C4 plants, BS chloroplasts are deficient in PSII, so the triose phosphate produced must be transported to M chloroplasts for reduction. Consistent with this role, the transcript of a maize TPT gene (APE2; GRMZM2G070605) was expressed with very high RPKM values of 2,069 and 1,551 in M and BS cells, respectively (Supplemental Data S3).
In C4 plants, pyruvate produced from malate decarboxylation in the BS chloroplasts must be returned to M chloroplasts for regeneration of PEP via PPDK with the input of ATP, and PEP is subsequently exported to the cytosol for carboxylation by PEPC. Earlier biochemical and molecular studies suggested that to accommodate these new transport functions in C4 plants, TPT has been adopted and modified for the exchange of phosphate and PEP (PPT; Flugge, 1999), and two types of light-dependent pyruvate transporter have been employed by the M chloroplasts for the uptake of pyruvate in a wide range of C4 plants, one sodium dependent (Ohnishi et al., 1990; Ohnishi and Kanai, 1990; Aoki et al., 1992) and one proton dependent (Aoki et al., 1992). The NADP-ME subtype C4 Flaveria spp. use the sodium/pyruvate symporter (Gowik et al., 2011), but maize, a NADP-ME subtype monocot, uses the proton/pyruvate symporter (Ohnishi et al., 1990; Ohnishi and Kanai, 1990; Aoki et al., 1992). The gene coding for the sodium-dependent pyruvate transporter BASS2, a bile acid:sodium symporter family protein 2, has been cloned recently from C4 Flaveria spp. (Furumoto et al., 2011), but the gene(s) coding for the proton-dependent one remains elusive. Transcripts of several maize PPTs were detected in this study. One of them (GRMZM2G174107) was highly enriched in M cells, whereas a second one (GRMZM2G066413) was moderately enriched in BS cells. Thus, the GRMZM2G174107 gene may specifically code for the exchanger of triose phosphate and PEP in the M chloroplasts of maize leaves. In addition, our transcriptome data showed that for the three MEP (for methyl erythritol phosphate) genes coding for the inner envelope transporter proteins, MEP3 (GRMZM2G138258) was expressed exclusively in BS cells (Ri = 0.99) at an extremely high level (RPKM = 2,734), GRMZM2G305851 was moderately expressed with a preference in M cells, whereas MEP2 (GRMZM2G077222) was equally expressed in both cell types at relatively low levels. The exact physiological roles of MEP transporters in maize have not been established, but all three MEP transporters have chloroplast-targeting sequences (by TargetP; http://www.cbs.dtu.dk/services/TargetP/) and transmembrane domains (by DAS; http://mendel.imp.ac.at/sat/DAS/DAS.html), which suggest that they are plastidial transporters. Since pyruvate is one of the two crucial substrates of the MEP pathway in plastids, we propose that the two MEP3 paralogs may code for the inner chloroplast envelope proteins responsible for pyruvate export from BS chloroplasts and import of pyruvate by M chloroplasts, respectively, whereas MEP2 may mainly function to supply pyruvate to the MEP pathway, as in C3 plants. In agreement with our data, Friso et al. (2010) suggested earlier that MEP2,4 and MEP3/4 may function as pyruvate transporters in M and BS chloroplasts, respectively, based on proteomic studies of isolated maize M and BS chloroplasts.
Although photorespiration is not apparent in C4 plants under ambient atmospheric conditions, measurable photorespiratory activity can be detected at low CO2 conditions (de Veau and Burris, 1989; Dai et al., 1993) and in young leaves (Dai et al., 1993, 1995). Consistent with the localization of Rubisco in the BS chloroplasts of C4 plants, the expression of genes coding for most of the key photorespiratory enzymes exhibited a BS specificity (Supplemental Data S3). High levels of transcript were detected in the BS cells for glycolate oxidase (GRMZM2G129246), Gly decarboxylase (GRMZM2G399183 and GRMZM2G104310), and Ser hydroxymethyltransferase (GRMZM2G135283), with Ri values ranging from 0.94 to 1.00. However, expression of two of the photorespiratory pathway enzymes, chloroplast phosphoglycolate phosphatase and peroxisomal NAD-hydroxypyruvate reductase, did not show strong BS preference, presumably due to their housekeeping roles in the organelles. Also, chloroplast is known to be capable of assimilating ammonium, a toxic compound released from Ser decarboxylation in mitochondria, through Glu synthetase and Gln synthase. In accordance, genes for Glu synthetase (GRMZM2G098290) and Gln synthase (GRMZM2G036609) were highly expressed in both cells. It is also notable that several paralogs of Ser hydroxymethyltransferase were expressed in maize, but only one of them (GRMZM2G135283) was exclusively expressed in BS cells, which could have been acquired during the evolution of C4 plants.
Development, Cell Wall, and Plasmodesmata Formation
Kranz leaf anatomy is essential for most C4 plants to concentrate CO2 around Rubisco in the chloroplasts of BS cells to suppress its oxygenase and the associated photorespiratory activity. Consequently, the BS cell wall in many C4 plants is thickened and specialized with a suberin layer to prevent CO2 leakage (Evans and Von Caemmerer, 1996; Leegood, 2002). Our data depict the expression of many cell wall-associated genes specifically expressed in BS cells, which include genes coding for cell wall-associated kinase (GRMZM2G135291 and GRMZM2G062471) and several highly expressed nodulin-type proteins (MtN3 and MtN21). Therefore, these genes may be related to the differentiation of C4 BS cell walls. In contrast, only one cell wall-related gene coding for expansin (GRMZM2G094990) is specifically expressed in M cells.
Intercellular transport of metabolites between M and BS cells (Evert et al., 1977; Robards and Lucas, 1990) and between the BS and the adjacent vascular parenchyma cells (Botha et al., 2000) in C4 leaves is mainly by diffusion through interconnecting plasmodesmata, which are concentrated along the cell wall between these cell types. Interestingly, our data reveal that the genes coding for plasmodesmata-located proteins (signaling receptor kinases), plasmodesmata callose-binding proteins (β-1,3-glucan hydrolases), and callose synthase are almost exclusively expressed in BS cells (Supplemental Data S3), suggesting that genes related to plasmodesmata development may be controlled by the regulatory network of BS cells. Callose, which differs from cellulose in consisting of a β-1,3-glucan chain, is known to be made by a few cell types at a specific stage during cell wall development, such as microspore and pollen. It is also ectopically deposited over the cell wall between the BS and vascular parenchyma cells in maize leaf minor veins, thereby occluding the plasmodesmata (Botha et al., 2000). Therefore, we speculate that a similar cell wall composition may be shared by nodule, microspore, pollen, xylary fiber, and C4 BS cells. C4 plants regulate the expression of these cell wall-related genes in an organ- and cell-specific manner to function in the CO2-concentrating mechanism.
The C4 physiology is an integrated syndrome of developmental, anatomical, cellular, and biochemical traits that must rely on regulatory networks that control the functional differentiation of M and BS cells. Our transcriptome profiling of the two photosynthetic cell types of mature maize leaves reveals the cell differentiation of gene expression at both regulatory and functional levels. To our knowledge for the first time, genes coding for the transporters of several C4 metabolites and the development of the BS cell wall have been identified that express in a cell-specific manner. Clearly, both cell types are specialized in different metabolic functions but are also coordinated to provide a high efficiency in carbon, mineral, and water use. In addition, many TF genes have been shown to express in a cell-specific manner. Thus, the comprehensive data sets from this study have laid a solid foundation for further study of the regulation of the expression and evolution of C4-specific and -related genes.
Although C4 plants represent only approximately 3% of plant species, they contribute to about 30% of terrestrial carbon fixation, and 60% of C4 plants are grasses, which include some of our most important food crops, such as maize, sugarcane (Saccharum officinarum), millet (Pennisetum americanum), and sorghum (Sorghum bicolor) of the NADP-ME subtype. Despite this scarcity, some of the most productive crops and noxious weeds on earth belong to the C4 plants, which make this photosynthetic property a desirable trait for introduction into the less productive C3 crops (Hibberd et al., 2008). However, at present, very little is known about the molecular regulatory networks underlying the development of Kranz leaf anatomy and the functional differentiation of its photosynthetic cells in C4 photosynthesis and other metabolism. A comprehensive understanding of the C4 syndrome is required before we can effectively transfer the C4 traits into C3 crops, such as rice (Oryza sativa), wheat (Triticum aestivum), and soybean (Glycine max). A thorough and systematic examination of the regulatory networks involved in C4 syndrome from comparative C3, C3-C4 intermediate, and C4 transcriptome study (Gowik and Westhoff, 2011) and from developmental and cellular transcriptome profiling of C4 leaves (Li et al., 2010; this study) should streamline this process.
Plant Growth Conditions and Leaf Sample Collection
Seeds of maize (Zea mays 'White Crystal'), a glutinous cultivar, were germinated and cultivated in the greenhouse in June 2011 under natural sunlight conditions (13–14 h) with a maximum photosynthetic photon flux density of 1,600 μmol m−2 s−1, day/night temperatures of 28°C to 32°C/25°C to 28°C, and 60% to 70% relative humidity. The second newly expanded leaves of 9-d-old plants were harvested around 9 am for isolation of M and BS cells in the laboratory and extraction of total RNA.
Enzymatic Isolation of M Protoplasts
MP were isolated from the mid section (5 cm) of newly matured second leaves by digesting leaf segments in a cell wall digestive medium, according to the procedures of Kanai and Edwards (1973), Sheen (1995), Markelz et al. (2003), Sawers et al. (2007), and Sharpe et al. (2011) with minor modifications. All solutions and glassware were sterilized either by filtration or by treatment with diethyl pyrocarbonate water followed by autoclave. The digestive medium, containing 1.5% (w/v) cellulose (Onozuka RS), 0.1% (w/v) macerozyme (Onozuka R-10), 20 mm MES (pH 5.8), 0.6 m sorbitol, 5 mm β-mercaptoethanol, 1 mm CaCl2, 0.1% bovine serum albumin, and 10 mm dithiothreitol (DTT), was prepared freshly from filter-sterilized stock solutions. MES buffer solution was preheated to 70°C for 5 min and cooled to room temperature before the addition of sorbitol and digestive enzymes. The medium was then heated to 55°C for 10 min to solubilize the enzymes as well as to inactivate DNase, and other components were added. Several leaf blades were stacked and cut perpendicularly to the long axis into 0.5- to 1-mm slices with sterilized razor blades and then quickly transferred into a 50-mL sterile beaker containing 20 mL of digestive medium. The medium with leaf slices was vacuum infiltrated twice and incubated at 23°C under low light (100 μmol m−2 s−1) for 3 h without shaking.
At the end of digestion, the mixture was first filtered through a 500-μm mesh, and the materials retained on the mesh were washed gently with 100 mL of sterile sorbitol wash medium (0.6 m sorbitol, 5 mm HEPES-KOH, pH 7.0, 100 mm β-mercaptoethanol, and 1 mm CaCl2) to release more MP. The filtrate was then filtered through an 80-μm nylon net to retain undigested tissue and BS, while MP (15–35 μm diameter) in the filtrate was collected by centrifugation at 300g for 5 min. The crude MP pellet was gently resuspended in 4 to 6 mL of 13% (w/v) dextran-Suc solution (13% dextran, 0.6 m Suc, 5 mm HEPES-KOH, pH 7.0, 100 mm β-mercaptoethanol, 1 mm CaCl2, and 10 mm DTT). For purification, the MP crude suspension was transferred to a 13-mL test tube and gently overlaid sequentially with 2 mL of 10.7% (w/v) dextran-Suc solution and 2 mL of sorbitol wash medium. The gradient was centrifuged at 300g in a swing-bucket rotor for 20 min, and pure MP was collected from the interphase between the sorbitol wash medium and 10.7% dextran-Suc solution and diluted in a small volume of sorbitol wash medium before isolation of RNA. The purity of isolated MP was examined with a light microscope.
Mechanical Isolation of BSS
To avoid damage to BS cells during enzymatic digestion, a mechanical method was adopted for rapid isolation of BS cells. The isolation medium contained 0.6 m sorbitol, 50 mm Tris-HCl (pH 8.0), 5 mm EDTA, 0.5% polyvinylpyrrolidone-10, 10 mm DTT, and 100 mm β-mercaptoethanol. Leaf slices, prepared as described above, were quickly added to 50 mL of cold BS cell isolation medium and homogenized twice (10 s each) at high speed in a Waring blender. After blending, the mixture was first filtered through a 500-μm mesh, the crude tissue retained on the 500-μm mesh was resuspended in another 50-mL isolation medium, and the blending and filtration processes were repeated. BS cells that passed through the 500-μm mesh were filtered and retained on an 80-μm nylon net. The purity of BS cells was monitored with a light microscope, and if deemed necessary, the collected BS cells were further washed with the cold isolation medium to remove attached M tissue and the epidermal tissue floating on top of the isolation medium. The whole isolation procedure was completed within 10 min.
Enzyme Assay and Immunoblot Analysis
The activities of NADP-ME and PEPC were assayed at 30°C using a spectrophotometer according to Kanai and Edwards (1973). The procedure of Murphy et al. (2007) was followed for immunoblot analysis using specific antibodies.
RNA Isolation, Library Preparation, and Deep Sequencing
Total RNA from isolated cells was extracted using Trizol reagent (Invitrogen) and purified using acid phenol-chloroform extraction. The quality of extracted RNA was examined by gel electrophoresis and by BioAnalyzer (Agilent).
Using the Illumina mRNA-seq kit, 10 to 15 μg of a total RNA sample was purified for poly(A) RNA and subjected to fragmentation for 3 min at 94°C. The mRNA fragments were then converted into single-stranded cDNA, and the ends were modified and ligated to the paired-end adaptors. The ligation products were size selected on agarose gels (independent slices of 250–350 and 350–450 bp), amplified by 15 cycles of PCR, cleaned up using Ampure beads (Beckman Agencourt), and examined by Qubit (Invitrogen) and BioAnalyzer High Sensitivity DNA chip (Agilent). Paired-end 2- × 120-nucleotide sequencing was performed on Illumina Genome Analyzer IIx, and two lanes of data were obtained for each cell type by sequencing two independently sized selected libraries. Approximately 177.4 million and 193.4 million reads were obtained from the M and BS RNA samples, respectively.
Deep-Sequencing Data Processing, Mapping, and Expression Level Calculation
We processed and mapped raw reads to the maize genome (B73) using the mapping tool Tophat version 1.3.3 (http://tophat.cbcb.umd.edu/) with default parameters (maximum allowed mismatches, two; maximum number of hits for a read, 10). Based on the APGv2 annotation downloaded from http://ftp.maizesequence.org/, we calculated the expression levels of each annotated gene in RPKM (Mortazavi et al., 2008) using the expression calculation tool Cufflinks version 1.2.1 (http://cufflinks.cbcb.umd.edu/).
Estimation of Cell Type Specificity
As described in "Results," to estimate the degree of differential expression of genes between the two cell types, we defined the degree of cell specificity of gene i as Ri = |mi – bi|/max(mi, bi), where mi and bi represent the RPKM of gene i in the M and BS RNA samples, respectively. Applying this measure to the transcriptome data, one can study the gene expression differences between the M and BS cells.
Estimation of Contamination Rates
We can use the transcriptome data to obtain an approximate estimate of the contamination rate in the M (or BS) cells isolated. Let c1 be the percentage contamination of the isolated M cells by BS cells. That is, c1 is the proportion of BS cells in the sample of M cells. For gene i, let xi and yi be the expected expression levels in M cells and in BS cells, respectively, under no contamination. Then, the expected expression level of gene i in the isolated M cells, denoted by mi, should be given by
Equation 1 cannot be solved because there are two unknowns, c1 and mi. However, if gene i is supposed not to be expressed in M cells, we can assume xi = 0 and obtain c1 = mi/yi.
Since using one gene to estimate c1 may not be reliable, we estimate c bywhere the summation is over the genes that are supposed to be expressed only in BS cells but not in M cells. We can take the estimate as an upper estimate because one or more of the genes used may not be absolutely only expressed in BS cells.
Similarly, let c2 be the rate of contamination of BS cells by M cells and let bi be the observed level of expression in the transcriptome data of the BS cells isolated. Then, we havewhere the summation is over the genes that are supposed to be expressed only in M cells but not in BS cells.
Functional Annotation
The identified cell type-enriched genes were annotated with biological functions and metabolic pathways by MapMan (Thimm et al., 2004; http://mapman.gabipd.org/). The TF information was downloaded from Plant TFDB version 2.0 (Zhang et al., 2011; http://planttfdb.cbi.pku.edu.cn/).
The read data have been submitted to the National Center for Biotechnology Information Short Read Archive under accession number SRP009063.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Micrographs of isolated M and BS cells.
Supplemental Figure S2. Immunoblot analysis in mature leaves and isolated M and BS cells (two preparations).
Supplemental Figure S3. Median depth of read-mapping coverage along the cDNA.
Supplemental Table S1. Activities of PEPC and NADP-ME in the whole leaf and in the isolated M and BS cells of maize.
Supplemental Table S2. Validation of cell type-specific gene expression.
Supplemental Table S3. List of quantitative reverse transcription-PCR primers, Universal Probe Library probe, and PCR efficiency.
Supplemental Table S4. RPKM of selected genes of vascular and epidermal tissues.
Supplemental Data S1. RPKM values of all genes with at least one read in one of the two transcriptomes.
Supplemental Data S2. List of genes with Ri > 0.99 in the two cell types.
Supplemental Data S3. Differential expression between M and BS cells of genes.
Supplemental Data S4. List of cell type-enriched TFs.
We thank Jen Sheen and Shin-Han Shiu for valuable comments and suggestions on the manuscript. The transcriptome sequencing was conducted by the High Throughput Sequencing Core, Academia Sinica, Taiwan.
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: Wen-Hsiung Li (whli{at}sinica.edu.tw).
↵1 This work was supported by Academia Sinica and the National Science Council, Taiwan (grant nos. NSC 99–2321–B–001–041–MY2 and 99B1–114).
↵[OA] Open Access articles can be viewed online without a subscription.
↵[W] The online version of this article contains Web-only data.
- mesophyll
- bundle sheath
- laser-capture microdissection
- mesophyll protoplasts
- bundle sheath strands
- malic enzyme
- phosphoenolpyruvate carboxylase
- reads per kilobase per million mapped reads
- Filtered Gene Set
- Working Gene Set
- phosphoenolpyruvate
- transcription factor
- triose phosphate/phosphate
- dithiothreitol
- cDNA
- complementary DNA
- Received July 17, 2012.
- Accepted July 23, 2012.
- Published July 24, 2012.


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This full page bead sheet was made by drawing by hand on an 8-1/2" by 11" sheet of paper with a green and yellow pen. Then it was scanned into the computer. It is in PDF format so it can be used on any computer. It can be used for bead sheets or any other paper craft! The sample beads are made with white 110lb card stock.
Spring Chaos

subset: commoncrawl


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hidden moon
Yesterday's Phenology: A chilly breeze, and cloudy. In the morning, fog, and frost on the grass and the bushes. The winter sky is owned by crows. This morning they were flying west to east, tree to tree, branch to branch, a crow parade, yakking it up the whole way. Out for a lengthy dog walk in the afternoon, the path a wet mat of brown leaves. Tonight it rains.
Song of the day: Gustav Holst, Neptune, the Mystic


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The result of the Israelite's unbelief of God led to their death in the wilderness; hence, the older generation never entered the promise land. In fact, God raised up another generation as the unbelieving generation died. Thus, (Heb 4:2) tells us "for unto us was the gospel preached, as well as unto them; but the word preached did not profit them, not being mixed with faith in them that heard it." Thus, they heard the word as Moses preached, but they didn't believe or receive the word. And many people who confess to be Christians today hear the word which is being preached on Sunday morning; however, they seem to have a hard time believing the teachings. Child of God it doesn't matter how well we sing or how good we look in our Sunday morning outfit; neither how beautiful your praise dance looks. However, the thing that matters the most is "Do you believe God?" If unbelief kept the Israelite's out of the promise land surely it will keep us out of our promise land which happens to be heaven. Let us mix the teachings of God's word with faith and bring forth a spiritual cake which we can present to God as a sweet smelling offering of our lives that we might be able to enter into the "Rest" of God. There is no sense in going to church Sunday after Sunday and walking in unbelief; we are simply wasting our time if we won't believe.
God Bless You.


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Rachael Ray Hard Enamel Nonstick 8-Quart Covered Pasta Pot, Green Gradient
$55.36 $79.99
Chili, pasta, corn on the cob - you name it - the Rachael Ray Hard Enamel Nonstick 8-Quart Covered Pasta Etc. Pot is the ideal stockpot for whatever is on the menu. The pot's innovative oval shape not only takes up less room on the cooktop, but makes it easier to boil lasagna and spaghetti noodles without breaking them - and a convenient pour spout helps pour out liquids. Combining superior performance, a fun contemporary color and durable materials, this pasta pot can handle everyone's favorite dishes. From Rachael's Green Tortilla Chili to her Spaghetti and Meatball Stoup, cooking at home has never been so satisfying. The pot's sturdy construction promotes even heating, helping to reduce hot spots that can burn foods, and the bold hues of the pot's two-tone enamel exterior show plenty of stylish kitchen personality. A durable nonstick interior provides superior food release and makes cleanup a breeze. Comfortable grippy handles are double riveted for extra strength and the shatter-resistant glass lid locks in heat and moisture. Oven safe to 350°F, this pasta pot goes great with many other items in Rachael's collections. Choose the versatile and uniquely shaped Rachael Ray Hard Enamel Nonstick 8-Quart Covered Pasta Etc. Pot whenever simple, delish food is on the menu. Color: Green.
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1019 Belvidere Rd
Waukegan, Il 60085
south east corner of Belvidere and Jackson
Belvidere Rd is also route #120
The Salmon Stop is east of the tollway and east of Rt. 41.
9 stop lights from tollway {94/294} 7 stop lights from 41.
Hours seasonal, please call first.
Phone number: 847-244-2525
Fishing hotline:847-244-9876
Accepting all credit cards and cash but sorry, no checks
The Salmon Stop offers:
Personal, courtious service, we will even take you out in the parking lot
and show you how to cast your new rod and reel if your not sure how to use it!
Rod, reel, and downrigger repair
Il and WI fishing and hunting licenses
A full line of live bait
Tackle for small lakes as well as a huge selection of the latest hot items for Lake Michigan.
They have rods and reels of all price ranges, lots of rod/reel combos, and something is always on sale!
Charter Info
If they don't have an item you want in stock, they will try to order
it for you.
Ever been to a big chain store where no one knows anything about what they are selling?? That won't happen here.


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Please read these important instructions carefully before proceeding:
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A valid "SELLER'S PERMIT" is required to purchase a space online.
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Seller(s) will not be allowed to sell without a valid "SELLER'S PERMIT" and there will be NO REFUND.
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Notice: The sale of food that can be consumed on property is not allowed. All produce vendors and vendors selling other consumables must have permits from the Health Department and approval from management before selling.
We do have a competition rule in the park.
You cannot sell within 3 spaces of a lease holder who sells the same time of merchandise you have. On the sales platform, the lease holders are represented by all of the Blue Boxes (Reserved). If you mouse over the space, you can see the vendor name and merchandise.
1. If you bought a space in advance, please arrive between 6:00a.m. – 7:15a.m. If you arrive after 7:30a.m. you will be charged a $10.00 late fee.
2. Spaces will be held until 7:30a.m. ONLY. After 7:30am management reserves the right to resell the spaces without a refund or raincheck to purchaser.
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2.5″ Softy Leather Guitar Strap XL – Black
The 'Softy' handmade from leather with padding and a suede back. This is soft in nature and padded making it a very comfortable guitar strap to use. The XL straps are approximately 6″ longer than the standard straps.
*Photograph colours may appear slightly different to actual product


text: Our custom wine glasses serve up your logo on two sides for no additional cost! Use a satin screen for that etched look, or your choice of imprint colors. The trendy 16.75 oz. Vina Stemless Red Wine Glass will be sure to turn heads at your next event! Promotional wine glasses make excellent customer gifts or employee appreciation gifts. Ask our knowledgeable sales people which style glass best fits your needs.


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Some of the most interesting beers in Brooklyn don't come from craft breweries or beer bars — they can be found fermenting in the borough's home kitchens and basements.
On April 12, the "Pride of Brooklyn Homebrew Festival" will return to Littlefield in Gowanus for its second year, offering 25 amateur brewers the opportunity to show off their finest creations — and local beer-lovers the chance to sample them.
"Craft beer is becoming more experimental and adventurous, and people are becoming more curious about how to make their own," said festival coordinator Casey Soloff. "There's a real interest among a certain segment of people who want to understand what goes into it and who want to make the type of beer they want to drink."
The best beers at the festival will be awarded prizes by both professional judges and the audience, but Soloff said the event is as much about community as it is competition.
"Brewers are overwhelmingly nice people," said Soloff, who has himself dabbled in home brewing. "People come here with a tasting glass, but it's really more like a hangout. The people who provide the beers are really passionate about them."
Last year's first-place winner said that concocting creative brews is the key to winning the judges' and drinkers' favors — and it is also the most rewarding part of the hobby.
"I love brewing because it's a creative, alchemical process that involves science, botany, history, principles of cooking, and experimentation," said Shannon Bowser. "You can make specific styles of beer that you want to drink that may be hard to find, or expensive to buy."
Bowser, a sculptor who lives in Philadelphia, took home the top prize in 2013 with a French saison called "Bel Soleil," and is hoping for a repeat performance this year with her persimmon gose, "Triumph of Spring."
But win or lose, Bowser said the greatest prize is just creating a new brew that friends and other beer geeks love.
"Home brewing builds a great community of like-minded people," she said. "You get a huge sense of accomplishment and pride when you brew a great batch that your fellow home brewers actually enjoy drinking."
"Pride of Brooklyn Homebrew Festival" at Littlefield [622 Degraw St. between Thirs and Fourth avenues in Gowanus, (718) 855–3388, www.little
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Hi Beauties! This is the new releases on .:TheBeautifulOnes:.
As some of costumers ask me for the boho patched pants in more sizes i re-made this beautiful pants for mesh bodies plus standard sizes plus fitted sizes.
Avaliable on store & marketplace.
Plus! Tomorrow only 55L promo at store! Taxi -> http://tinyurl.com/storetbo
♥ Babs.


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Make It: DIY Tiki Torches from Upcycled Glass Bottles
Turn sad, empty bottles into festive outdoor mood lighting perfect for chilly fall soirees! It's super easy -- here's how.
Tiki torch wicks, glass bottles (empty due to festivities past -- and cleaned out, of course), and tiki torch fluid are all you need for this simple project from Sugar and Charm. Once you've gathered your supplies, check out their tutorial for piecing everything together!
[via ManMade]


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March came in like a lion here in my corner of cyber space… fabric messes and sorting it by color. I don't know if the baskets are a good choice, but it is what I had..And the mail person brought me the invitation to my one and only Grand daughter's wedding. She is over 25 so I had given up on her meeting "that guy".
Well it happened and on the 24th I was down in Texas at her wedding.
That's my son with the matching hair… It was HIS daughter that started this whole thing of us dressing up and watching / witnessing their vows. My grandson is just 17 and is a very tall dude. Of course all the men from my side of the family are over 6 feet.. And the grand paw's side of the family is also very tall….6 foot plus!! He is handling his height very well.. and took very good care of this Granny.


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Love Birds Necklace
These gorgeous little lovebird necklace is made in fine eco-friendly recycled silver and has been aged slightly to bring out the detail.
Can be personalised with a special date or set of initials.
Comes on a 16" Sterling Silver necklace
Matching cufflinks available making a perfect wedding gift for the bride and groom


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Clougher calling! Clougher calling!
Deer Jelly,what exquisitive joy to heer your strong, barry-tone voice waft over the rolling prairies and deep ravines of Co Tyrone. I thank you from the bottom of my hart for standing in for Gerry Anderson. And my sun, Bon Jovi thanks you from his bottom too.
I am sure Jelly that you are compes-mentos of the fact that Gerry Anderson is running round Knew York, wearing a very short simmet and a wee pear of blew nickers. I wood be the last person on earth too say anything dee-ogg-raty about Gerry, but I can't help but feel he left it a bit late to start acting the Master McGraw. A man in the twilight years of his life should be dozing in front of the fire and sucking champ through a straw.
Your golfing handicap, Mr Coyle is also in Knew York. Walking about like a culshie with his mouth hanging open. Staring up at big, tall buildings like a boy who was never out of the house and shouting, "Hello there! How's it going?" to everyone he meets. What must the American's think of him Jelly? Walking about like Forest Gump with a green gansey on him and his name and address pinned to his chest.
I kan't sea any inward investment coming from this ill-fated, ill-timed, puke retching trip!
How is you Jelly? Us, me and Bon Jovi are as happy as Alasdair McDonnell, in a dimly lit room. A knew, thrusting, elequent leader who kan't read with the lights on-just what the SDLP was crying out for. Wait 'till HE hits America! Bon Jovi, arrayed in "Joe Bloggs" dungarees, sends his love. As do I, arrayed in hob-nailed boots, tartan, drindle skirt, puce blouse and a laurel wreath of germaniums in my hare. If the good Lord's willing and the creeks don't rise, I may send you another epistile before the weak end.
It just remains for me to sign off with a rousing , "Come On Yeh Boy Yeh and Keep Her Lit!!!!! Rosie Ryan xxx
Tuesday, 8 November 2011
A Culshie In New York.
Posted by Rosie Ryan at 02:25
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The mockingbird was apprehensive at first but is a regular visitor now. It favors the peanut butter cups but also eats the homemade suet.
It is so hard to believe that another year has ended. Honestly, I was glad to see that one go! I look forward to 2013 and I am trusting that it will be a better year for my family!
I am going to challenge myself for 2013 and try to post at least one picture a day. It may be an old picture I've taken and never posted, it may just be something I like, it may or may not include text. One thing I won't do is create more stress for myself, so if it becomes too much of a burden or chore I will stop.I need the challenge so that I will get back into the habit of regular blogging and improve my photography. Working has put both on the back burner and I want to change that.
I hope 2013 will be a great year for each of you!


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'You turned my life upside down:' sexual assault victim watches her assailant avoid jail time
In a story that has become ubiquitous, another male student avoided jail time after pleading guilty to sexually assaulting a student on the Iowa State campus. Patrick Whetstone, 21, avoided jail time for the 2014 assault, but will be required to register as a sex offender for ten years as part of a plea agreement. He also agreed to two years of probation. Judge James McGlynn agreed to the plea deal.
The victim told the court that she had felt ill and then fallen asleep after drinking on March 30, 2014. She woke up to find Whetstone assaulting her. The judge said that he believes the "sentence will protect the community" from Whetstone. Whetstone will also have to submit to evaluation and enter counseling if it is ordered. Whetstone will also have to turn over a DNA sample, and the judge said he believes that the perpetrator can be rehabilitated.
The victim, who was 19 at the time and who is going under the pseudonym "Jane Doe," is suing Iowa State University for its "botched response" to the assault, which led to the victim dropping out of school. Even though the victim reported the incident to Iowa State campus police right away, Whetstone was not charged for six months. The victim dropped out for the rest of that spring semester. When she returned to school in the fall, her new housing assignment was a block from where Whetstone lived, which caused her to run into him nearly every day.
Iowa State is one of 90 colleges currently being investigated by the federal government for its handling of sexual assault charges on its campus. Failure to provide a safe environment for students is a violation of Title IX, the 1972 statute that requires colleges that receive federal funds to provide equal accommodations and services to students regardless of gender.
Station KCCI reported that, prior to sentencing, the victim spoke.
"The woman who was sexually assaulted told her assailant that he changed her life forever.
Speaking in an Iowa courtroom, the 21-year-old woman told Whetstone that the 2014 assault "turned my life upside down" and shattered her college career.
She told Whetstone that "you touched my soul in a way that haunts me in my sleep." She spoke of having panic attacks and losing friends who didn't believe her.
The woman told one of Whetstone's attorneys that she's never been so "verbally disrespected by anybody in my entire life."


text: This holiday season, House of Raeford Farms locations have made a point to spread holiday cheer and give back to their communities. We are proud of our employees in South Carolina, North Carolina, Georgia and Louisiana for embodying House of Raeford Farms FLOCK values and helping those less fortunate. FLOCK donated nearly 50,000 pounds of… Read More


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There was something oddly familiar about Alex Rodriguez's Tuesday press conference on his steroid usage. I suddenly had a flashback to Eliot Spitzer and his wife Silda Wall nearly a year ago.
Though A-Rod's teammates sat together in the audience rather than right next to the embattled superstar like Silda did with Spitzer when he admitted to cheating with a prostitute the basic message conveyed in two similar settings was the same: The superstar has screwed up royally.
He had (using different senses of the word) "cheated."
But, in the case of Silda Wall, she chooses to put her personal loyalty and evident marriage vows over the feelings of betrayal that she must undoubtedly feel. Meanwhile A-Rod's teammates, regardless of how they must feel about the fact that Mr. Drama Player completely distracting everyone from what should be an exuberant spring training -- are obligated by team loyalty to "show up" at this press conference.
What is it about "loyalty" that makes it such a harsh taskmaster?
U.S. & World
Stories that affect your life across the U.S. and around the world.
In addition to Eliot and Silda, there's also Bill and Hillary -- not to mention Bill's entire administration (including VP Al Gore). Many of the reasons are personal and private that cause one individual to stay with another, even though, in so doing, the innocent party doesn't just agree to overlook the sins of the offender, the innocent essentially takes on those transgressions.
For a wife, like Silda Wall Spitzer, that means she has to take the abuse of the many observers who condemn her for literally "standing by her man" during a humiliating press conference -- and then, apparently, deciding not to divorce him afterwards.
In the political drama of ten years ago, arguably, Hillary Clinton's decision worked out for her, as she ended up a U.S. senator, presidential candidate and current Secretary of State. On the other hand, Al Gore's loyalty arguably cost him the presidency, losing to a candidate who pointedly promised to restore "honor and dignity" to the White House.
And now, we have A-Rod, who has admitted his "infidelity" to the game, by allowing steroids to be injected into his arm -- supposedly just during the a three-year period. But, as with the other cheaters, the cost of loyalty may be a steep one. Shortstop Derek Jeter -- who was at the press conference to "support" Rodriguez on Tuesday -- came out Wednesday to remind reporters that not all players were using performance enhancing drugs. It was (in his view) just a small minority.
No sooner did he go on record with that than he was the target of a pretty strong column asking why he hadn't spoken out on steroids before. Now, this was rather amazing, given how Jeter has been the golden boy of New York baseball for a dozen years. Yet, he is now being seen critically because he is trying to 1) support his teammate and 2) speak out for the broader integrity of the game.
For this, he gets slapped down.
Guess that's what you get for trying to be too loyal to both your teammate and the game.
Robert A. George is New York writer who dabbles in stand-up comedy. He blogs at Ragged Thots.


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We always need help finding families and raising money. If you know of a family that is in need please submit an application for them here on the website.
Another way to help is to donate shed antlers or do an auction on your social media. You can auction off an antler or item and have the winner send the money here on our website. We appreciate any help in this matter.
100%. The only expenses sheds for santa has is advertisement and the cost of wrapping and delivering the presents.
We will be posting here on the website and social media of the meeting date and place, feel free to come help.
Yes, please look for the family application here on our website.
Simply post a picture on your social media and state what the money will be going towards. You will be responsible for the shipping as well so keep that In mind. Most antlers cost $25-70 to ship.


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- Tickets >
- Concerts Tickets >
- Other Tickets >
- Southern Fried Chicks Tickets
Southern Fried Chicks Tickets
Get ready for Southern Fried Chicks, all the way live. Southern Fried Chicks is playing this year in many cities . Buy premium Southern Fried Chicks tickets and experience the thrill of a live concert with Southern Fried Chicks at affordable prices.
Related Cities
Southern Fried Chicks News
Family life can be stinkin' hilarious
� ? ?Comic year and stars.
March 2011
Television movies for the week of March 27
4 pm (c c) � ? � "white chicks".
March 2011
HOT DISH: Jennette McCurdy: From iCarly to iCountry
Author of Cookbook Hazel.
March 2011
What's happening
Music by Beijing.
March 2011
Last Night: Sugarland At RodeoHouston
Perhaps somewhat risky step, but it worked and we.
March 2011
Ticket Buzz:


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Aftab Earrings
Request a price ₹ 9,850
This is one of the new pieces inspired by wooden ornaments which are a part of architecture in Italian palaces.
A star may be a sun in its own solar system but to us it is visible only by the light of the moon. It is a perfect metaphor for a balance between the masculine and the feminine, of having the power and yet allowing another to shine in their own space.
Material : Hand engraved brass with gold plating and pearls.Size : 10 cm in length.
The jewellery comes in a travel-friendly fabric pouch & our beautiful wood keepsake box, along with a note about the collection, care card, and a personal note from the designer. If this is a gift do let us know the name of the person you'd like Eina to address the note to. We can also include your personalised message, which you can write in the "Notes to Designer" section on checkout.
14 cm in length and roughly 5.5 cm at it's widest.
Here is a mix of our classics and a few new pieces in gold and pearl under the masthead of The Sun & The Moon. The combination of gold and white is a classic pairing which reminds us of complementary equals - the sun and the moon, feminine and masculine, strength and kindness, action and meditation, spirituality and magic. These pieces are drawn from different collections, and are a mix of motifs that serve us perfectly throughout the year. Some of the new motifs are from architecture that moved me during my travels. The soft gracefulness of gold and pearls is an expression for those of us who know our power, and choose to express it through a passionate desire to love and protect every single living being who shares this life with us.
Shipping & Returns
We deliver worldwide with free shipping within India. We hand craft your order with love and passion, and allow you the option to co-create them by choosing between stones, textures, plating options, charms, chain lengths, bangle or ring sizes. Making these pieces to your specification will take us anywhere between 2 to 10 working days (12 to 14 working days in case of custom made jewellery), after which they will be shipped out to you. We appreciate your patience with the handcrafting process and we promise that the jewellery will make up for the wait! For urgent deliveries on special request, please contact us before placing your order, at +91 9831299125.Click here for more information.


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Last 3 Edits
- May18th, 2016. Page created, General info.
When you translate, your old self ceases to exist and a new one is formed. It is fully within the context on the new recursion/setting, as if the other did not exist. So everything must be in 'context' with your current whereabouts. (Only your Descriptor and Type remain constant, but some features might need explaining in 'context').
For a solid summary, read the 1st column of page 125 of The Strange Core Rulebook.
To summarise what this means.
Your Focus must suit the reality you are in. No Soars Through the Sky on Prime Earth. I have a list of appropriate Foci on each of the 'Worlds Visited pages. If you have one that can exist in the recursion you have come from (usually Earth Prime) and the one you have entered, you may 'drag' your focus with you. (You don't have to. You always have the option of changing and being someone different).
Your new, reformed self comes equipped for the new recursion as if you had not been to the old. In essence, the old equipment does NOT exist. You can re-equip for the new recursion. Easiest way to do so is roughly convert your equipment, but don't feel you have to convert EVERY piece. It really does not matter if you add some mundane, cheap equipment either. (This is especially the case for your transition back to Prime Earth, as the Estate can equip you with basics).
We do need to keep a 'similar level of wealth' for your PC b/w recursions though. So currency converts; eg, from gps to $. Obviously in the case of Earth it is $ (though you probably had no $ on Blighted World).
I would simply go by your Type & Focus. Type states how many items of each expense level you have. (Focus may modify this of course). So keep an eye on your Type when deciding whether you have a car, mobile, laptop etc.
These DO translate, BUT their form must change to work in the context of your new recursion. This could be hard to impossible for some, but try and think of something similar or possible, or lose the cypher.
This may also change if there are non-humans on other recursions. For ease of use I suggest we just use the Combining Race/Species with Descriptor idea I have on our Cypher House Rules Page. However, if you prefer, as in the Core rules, race/species can simply replace your Descriptor (though I think this would be confusing over time). The setting pages list which races/species are available for PCs.
Gender & Appearance.
Yes, this may even change (just like race/species).
Some skills are obviously unique to certain recursions. These also change to be relevant in your current recursion/world. This is especially true of Knowledge skills. So knowledge of bio-engineering might not be appropriate in a fantasy recursion for eg. You can change it to something similar and in context.
The important thing to remember is that you are not 'teleporting', you are 'translating'; you change to 'fit in' to the context of your new recursion. Hope that helps for PCs.


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It is ironic but two unfortunate terror strikes have come as the opportunity cost to show verbal restraint in India-Pakistan ties – something that should be identified as a 'real time real' development in India-Pakistan thaws.
First it was Pathankot terror strike in India – on Pathankot air base – near the Pakistan border in Indian state of Punjab. The terror strike that began early in the morning of January 1 when, like the whole world, India, too was welcoming the New Year and went on for quite some time. The attack had clear Pakistani imprints (and footprints, literally). Though the attack did not cause much damage, it dragged on for hours and caused irreparable loss with seven Indian soldiers martyred.
And first time in the known history, at least in the recent times, Pakistan reacted sincerely to the leads points its hands there – even if it was under pressure to do so – under US pressure – and under the pressure of Narendra Modi's surprise Lahore stopover and diplomacy that ensued.
Pakistan condemned the attack and its prime minister's office released a statement saying Pakistan was seriously working on the leads provided by India and several Jaish-eMuhamman operatives were arrested. The statement also said that a Special Investigation Team was constituted for comprehensive probe. JeM is a Pakistan based terror group that is behind this attack on Pathankot. Its chief Masood Azhar, who was in Indian jail, was swapped in 1999 Kandahar hijacking incident.
It's true that we cannot expect much from Pakistan. That is visible from its flipflops on Masood Azhar's arrest.
But we need to appreciate the restraint shown. Pakistan's ruling establishment and even its military have tried to look sincere this time. Yesterday, a report came that Pakistan had assured the US of no doublespeak in Pathankot terror probe on its soil.
Let's give Pakistan some time to see what it comes with – as it time and again – after the Pathankot air base attack incident – has reiterated that it will reveal something concrete once the probe is finished.
India bashing was regular in Pakistani media – as usual -as expected – as the stories planted on crackdown on JeM and Masood Azhar's arrest.
The second indicator came today – with terror strike at Bacha Khan University in Charsadda district in Pakistan's Khyber Pakhtunkhwa province (KP). Around 25 people were killed when terrorists attacked the University founded in 2012 in the name of Gandhian Khan Abdul Gaffar Khan or Frontier Gandhi.
Like has been the practice in Pakistan, blaming India blindly for every terror strike, was not the case today.
No one in Pakistan's ruling establishment, including its civilian government and its military, rushed to blame India today – and that was very sincere.
In fact Nawaz Sharif responded sensitively to Narendra Modi's message in this crisis hour saying "this incident is yet another reminder that terrorism is our common enemy and a cooperative approach is essential to eliminate it from the region."
Yes, as usual, a section of Pakistani media has been on an overdrive to prove Indian hand in Bacha Khan University attack – blaming Indian Embassy in Kabul and providing some phone numbers that were used – but that looks more like a 'tit for tat' – with India's actionable evident provided to Pakistan in Pathankot attack incident. Hope, Pakistan government it's all powerful army would not go into that rhetoric.
Pakistan's ruling establishment knows that linking India to attacks in Pakistan is nothing but to appease the elements in its domestic politics – and the time is not opportune for that. Promoting unrest in India has been a state policy for Pakistan while India has always been a responsible democracy.
©/IPR: Santosh Chaubey – https://santoshchaubey.wordpress.com/


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The Apollo 11 Lunar Landing
There may be few -- if any -- TV moments that fired the world's imagination like the televised broadcast the night of July 20, 1969, when astronauts Neil Armstrong, Michael Collins and Ed "Buzz" Aldrin landed on the moon. For the first time in history, humans set foot on another celestial body.
And the world was there with them.
NASA pilots and astronauts, and their counterparts in the USSR, had already accomplished a number of feats: breaking the sound barrier, flying into space and performing work in orbit. But the lunar landing was one of the most significant undertakings in the space race, and NASA wanted to make sure that the event was broadcast to as wide an audience as possible.
The effect was striking. An entire generation of Americans -- the Baby Boomers -- marked the late-night broadcast as a pivotal moment in their childhoods. If America could put a man on the moon, they reasoned, anything would be possible. That optimism has marked that generation for decades, and it stands as living proof of the power of the live broadcast.


text: Abonnez-vous à ma lettre This thingy has to be empti pwieshh 18 août, 2017 Chatbots weekly digest - Issue #49 Revue Marketing Conversationnel pour le E-commerce 18 août · Numéro #49 · Consulter en ligne Botfuel sélectionne pour vous le meilleur des articles en français et en anglais sur l'expérience client dans le E-commerce, l'IA, le Marketing Conversationnel et les Assistants Shopping. https://www.botfuel.io/fr/ Analysis The Evolution and Future of AI in Conversational Commerce and Customer Experience www.forbes.com – Partager You can credit IBM's Watson for creating a platform for artificial intelligence integration into a multitude of business and customer-facing applications. You can thank Facebook for all the buzz about chatbots and the gold rush of developers and venture capital creating an onslaught of bot companies. You can laud Apple's Siri and Amazon's Echo for implementing intelligence into everyday devices and bringing to life voice as the new user interface. However, the truth is we are only just starting to realise the potential of artificial intelligence and one of the most promising applications for AI to make a more than welcome impact is in customer experience. It can be debated that for all of the good technology has offered businesses over the last several decades, it has also comprised key elements of the customer experience. Call centres, websites, email, apps, et al, have allowed companies to scale and automate customer engagement while also introducing process efficiencies and cost management systems. But that also comes at a cost. Over the years, many customers have complained that these very processes and systems are the very things that hinder or undermine satisfaction. How bots will change the way we shop venturebeat.com – Partager Consumers spend an average of five hours per week making online purchases; over 96 percent of Americans shop online, allocating an average 36 percent of their shopping budgets to ecommerce. With the shift to online shopping, retailers need to figure out how to connect with their consumers and still create a meaningful experience in order to continue to drive store visits. AI-powered assistants can also infuse personality into the shopping experience. As traditional brick-and-mortar shopping transitions online, consumers are losing out on the personal touch of store associates and friendly faces. Instead, they search a website on their own, often having to evaluate their purchases without a helping hand. Having a chatbot or AI-powered assistant on a website recreates the store experience for shoppers right in their living room. A lesson from the overreaction to the Facebook AI controversy venturebeat.com – Partager What's really happening? For starters, there is a fear of the unknown. It sounds suspicious at face value when you hear that bots did anything on their own. The truth is a little more granular than that. Adaptive intelligence might seem like it involves code that suddenly starts running amok, but even when a bot appears to "adapt," it is still following subroutines that are highly choreographed. Chatbot or human? There's only one thing that matters venturebeat.com – Partager All we want is results As long as we reach the desired outcome of our conversation, we don't care. Robot? Fine. Human? Fine. Hybrid? Sure. Both of these studies draw attention to the efficiency aspect of chatbots. Most consumers have not yet interacted with chatbots (about 75 percent in the U.K. have not), and yet many have high expectations for them. Though consumers do not know exactly how chatbots work, they know they are robots that are available 24/7 and are there to answer questions. This is more than enough information for many to make up their minds. We want answers and we want them now, so chatbots seem to be the way forward. Design makes AI smarter uxdesign.cc – Partager Collecting human input and data is crucial to achieving artificial general intelligence. We need to design experiences that incentivize engagement that improves the AI. At times, that means prioritizing AI over users, putting more effort on the users to teach the AI. 😮 Designers help artificial intelligence make meaning of user inputs and the implicit signals associated with those inputs. On top of the AI understanding the whats, we decipher the whys from the users' point of view. Bot building Want a better bot UX? Think like a game designer chatbotsmagazine.com – Partager How many times have you gotten frustrated with Siri for not understanding what you said? How many times have you wanted to punch the screen after it kept telling you that your input was wrong? We've all been there, and this can be one of the biggest pitfalls to achieving a delightful user experience. As botmakers, our job is to lead the users toward their goals, not leave them confused. As it turns out, the world of gaming can offer some time-tested insights on how to interact with users. In particular, we're going to look at the Interactive Fiction (IF) community, and how the strategies employed in early computer games can help you build better UX. Should You Leave Strategic Flaws in Your Bots? chatbotsmagazine.com – Partager To communicate and interact with each other, humans use various social cues in addition to other behaviors. For example, if you're talking to someone who doesn't agree or feels very strongly about a subject, you can tell by their body language and demeanor — even if what they are actually saying tells a different story. It's similar to looking for tells when a person is lying. The problem with modern robots and AI systems is they cannot read or discern this behavior. This makes them inefficient, in a way, and unable to participate on a social level. But if what a recent study shows is true, future robots may be able to decode or translate human social signals to understand an error or problem and react accordingly. How is this possible? Believe it or not, through the reactions humans have to a robot that makes mistakes. Interesting bots Chatbot helps students choose courses www.bbc.com – Partager The university's head of digital experience and engagement, Dougal Scaife, said: "We know that our prospective students already use lots of messaging software for communicating with their friends, such as Snapchat, WhatsApp, as well as texting, so developing a chatbot was a natural evolution in order to engage with our prospective students in a medium that is ubiquitous, familiar, and comfortable for them." Events Meetup Chatbots Paris⎪Chatbots Demo Night ⎪October 4, 2017 www.meetup.com – Partager 6 chatbots français sélectionnés pitcheront et présenteront des démos devant un panel d'experts des chatbots et de l'intelligence artificielle. Soumettez-nous vos projets ! AI With The Best online conference for developers⎪October 14-15, 2017 ai.withthebest.com – Partager AI With The Best is an online developer conference that'll connect students/developers/tech lovers with CEOs, CTOs, and researchers. During October 14-15, attendees will watch disruptive tech talks, schedule 1-on-1's with speakers, and accept challenges by speakers on our platform. There will be over 300 speakers from companies like Google Brain, Apple, Microsoft, Symantec, and more. Professors from top Computer Science universities will also be joining us. The biggest online developer conference gives you the possibility to engage and have private chats with your favorite speakers. Use BOTFUEL30 to receive 30% off conference tickets! Avez-vous aimé ce numéro ? Si vous ne voulez plus recevoir les prochaines mises à jour, vous pouvez vous désabonner ici. Si on vous a fait suivre cette lettre d'information et que vous l'aimez, vous pouvez vous y abonner ici. Propulsé par Revue Partager Share to Twitter Share to Facebook Abonnez-vous à ma lettre This thingy has to be empti pwieshh


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Reliable And Affordable Specialist Window Installers North London
A beautiful home needs inviting people, healthy environment and an attractive decor. Well designed doors and windows add character to […]
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