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Original Research ARTICLE
Genome-Wide Analysis of the Musa WRKY Gene Family: Evolution and Differential Expression during Development and Stress
- 1Council of Scientific and Industrial Research-National Botanical Research Institute, Lucknow, India
- 2Academy of Scientific and Innovative Research, New Delhi, India
The WRKY gene family plays an important role in the development and stress responses in plants. As information is not available on the WRKY gene family in Musa species, genome-wide analysis has been carried out in this study using available genomic information from two species, Musa acuminata and Musa balbisiana. Analysis identified 147 and 132 members of the WRKY gene family in M. acuminata and M. balbisiana, respectively. Evolutionary analysis suggests that the WRKY gene family expanded much before the speciation in both the species. Most of the orthologs retained in two species were from the γ duplication event which occurred prior to α and β genome-wide duplication (GWD) events. Analysis also suggests that subtle changes in nucleotide sequences during the course of evolution have led to the development of new motifs which might be involved in neo-functionalization of different WRKY members in two species. Expression and cis-regulatory motif analysis suggest possible involvement of Group II and Group III WRKY members during various stresses and growth/development including fruit ripening process respectively.
Transcription factors bind to the specific DNA motifs, regulate gene expression and control various signaling and regulatory networks involved in the proper growth and development as well as stress response in an organism. Out of numerous transcription factor gene families, WRKY gene family is known to be involved in various diverse processes in the plants from germination to senescence (Eulgem et al., 2000; Miao et al., 2007; Niu et al., 2012). Since the discovery of the first member of this gene family about 20 years ago, a number of members have been cloned and characterized from many plant species. In addition, with the advent of whole genome sequencing, the complete WRKY gene family has been analyzed in cotton (Dou et al., 2014), grape (Guo et al., 2014), populus (He et al., 2012), and many other plants.
This transcription factor gene family was named WRKY due to presence of highly conserved sequence WRKYGQK at N-terminus of the proteins (Rushton et al., 1995). In addition, members of this family also contain a conserved metal chelating zinc finger motif (C-X4–5-C-X22–23-H-X-H or C-X5–8-C-X25–28-H-X1–2-C) at the C-terminus of the WRKY motif. Members of this gene family regulate expression of genes through binding to the specific DNA sequence, W-box [(C/T)TGAC(T/C)], present in the promoter region of the target genes. In addition to the W box, WRKY domain also binds to a sugar responsive element, SURE, to regulate transcriptional expression (Sun et al., 2003).
In spite of the presence of highly conserved DNA binding domain, the binding of WRKY proteins to target genes varies due to the presence of variable number of WRKY domains and different pattern of zinc finger motifs (Rushton et al., 1995). Based on these differences, the WRKY genes are divided into three main groups (Eulgem et al., 2000). Group I and Group II WRKY members contain two and one WRKY domains respectively however; members from both the groups contain one zinc finger motif pattern of C2-H2 (C–X4–5–C–X22–23–H–X1–H). Group III WRKY genes contain one WRKY domain but vary in the presence of a different zinc finger motif pattern of C2-HC (C–X7–C–X23–H–X1–C). Based on the presence of different primary amino acid sequence, Group II WRKY genes are further divided into five subgroups (IIa, IIb, IIc, IId, and IIe; Eulgem et al., 2000).
Differential expression of the members of WRKY gene family and their involvement in various regulatory processes associated with biotic and abiotic stresses has been studied in various plants (Eulgem et al., 2000). In rice, OsWRKY71 (Liu et al., 2007), OsWRKY3 (Zhang et al., 2008), OsWRKY 45-1, and OsWRKY45-2 (Tao et al., 2011) have been shown to be induced during bacterial pathogen attack. Similarly, WRKY genes like AtWRKY8 (Chen et al., 2010), AtWRKY3 (Zheng et al., 2006), AtWRKY25 (Zheng et al., 2007), AtWRKY11, and AtWRKY17 (Journot-Catalino et al., 2006) are down-regulated under bacterial pathogen attack. Studies also suggest that OsWRKY11 (Wu et al., 2009), HvWRKY38 (Xiong, 2010) TaWRKY2, and TaWRKY19 (Niu et al., 2012) provide resistance to plants under drought stress. Members of the WRKY gene families are also known to regulate synthesis of the specific metabolites in response to abiotic stresses. Studies elucidated involvement of OsWRKY11 in enhancing raffinose accumulation required for tolerance to plant against desiccation (Wu et al., 2009). In addition to the involvement to stress response and adaptation, involvement of WRKY genes has been demonstrated in the regulation of genes responsible for the proper growth and development of the plant. In Arabidopsis, studies suggest involvement of AtWRKY6, AtWRKY22 and AtWRKY53 in regulating senescence process (Robatzek and Somssich, 2002; Zhou et al., 2011; Miao et al., 2007). In rice, over-expression of OsWRKY89 leads to reduction in the growth and internode length (Wang et al., 2007). OsWRKY72 delays seed germination under normal conditions (Yu et al., 2010) and AtWRKY7 was found to have a calmodulin (CaM)-binding domain (CaMBD) thus interacts with CaM (Park et al., 2005). Various studies suggest that alkaloid biosynthesis is regulated by members of WRKY gene family (Kato et al., 2007; Mishra et al., 2013; Agarwal et al., 2015). In addition to alkaloid, biosynthesis of the sesquiterpenes in cotton has also been demonstrated to be regulated by WRKY genes (Xu et al., 2004).
Therefore, it seems that WRKY genes play important role during plant growth, development and stress response. Studies suggest that during fruit ripening, expression of a number of genes related to stress response is enhanced (Kesari et al., 2007). However, involvement of WRKY gene family in fruit ripening has not been studied. The identification and functional characterization of the WRKY gene family from fruit crops will provide an insight into the regulatory aspects of biochemical and physiological processes operating during fruit ripening as well as response to various environmental stresses. Recently, the genome of two banana species; Musa acuminata (A genome) and Musa balbisiana (B genome) have been completely established (D'Hont et al., 2012; Davey et al., 2013). In this study, we have used these genomic resources to identify members of the WRKY gene family and correlated their expression with plant growth/development and stress response. Gene structure organizations and genome duplication events have been studied in detail using WRKY gene family from M. acuminata and M. balbisiana genomes. Their ortholog and homolog pairs have also been identified and their expression analysis was performed using available transcriptome datasets to identify involvement of specific WRKY gene family members in different processes.
Materials and Methods
Identification and Multiple Sequence Alignment
Protein coding (CDS) and whole genome sequences of two banana species, M. acuminata and M. balbisiana, were downloaded from banana genome database (http://banana-genome.cirad.fr). Hmmer-2.3.2 was used against the protein sequences of both the banana genomes to search the WRKY transcription factor encoding genes using plant Transcription Factor database (http://planttfdb.cbi.pku.edu.cn/; Jin et al., 2014) with E-value ≤ 10−5. All the identified WRKY genes were aligned separately against each other using multiple sequence alignment tool ClustalX2. The 60 amino acid conserved WRKY domain region was extracted from the sequences of identified proteins from M. acuminata and M. balbisiana. The WRKY genes which contain two WRKY domains, the domains were named as "C" or "N" depending on presence of domain at the C- or N-termini of the proteins respectively.
Phylogenetic Analysis
Due to the variable lengths of the complete protein sequences of the WRKY members, WRKY domains extracted from the identified proteins were used to draw the phylogenetic tree. Arabidopsis thaliana (85), Oryza sativa (121), Vitis vinifera (71), Zea mays (185) (Wei et al., 2012; Guo et al., 2014) and those identified from M. acuminata as well as M. balbisiana were used to construct the phylogenetic tree, making it a total of 778 non-redundant WRKY domains. The unrooted phylogenetic tree was generated using Maximum Likelihood algorithm with 1000 bootstrap value using JTT algorithm in MEGA 5.2 (Tamura et al., 2011). The phylogenetic tree generated was divided into the different groups on the basis of Arabidopsis and rice annotations and classification.
Gene Structure Analysis
The gene structure information for each identified WRKY gene was extracted from the general feature format file of their respective genome and the gene structure images were generated using GSDS (Gene Structure Display Server; http://gsds.cbi.pku.edu.cn/; Hu et al., 2015) online server. The chromosomal images showing location of WRKY genes were generated using the MapChart program (Voorrips, 2002).
Identification of Motifs and Tandem Genome Duplication
The conserve motif patterns were generated for the identified WRKY genes using MEME server (Bailey et al., 2009) with zoops models of minimum and maximum weight of 6 and 60 amino acid residues, respectively. These motifs were annotated using The Eukaryotic Linear Motif (ELM; http://elm.eu.org/) resource (Dinkel et al., 2014). Whole genome duplications were analyzed within the M. acuminata and M. balbisiana genomes using Musa ancestral blocks available at Plant Genome Duplication Database (PGDD; http://chibba.agtec.uga.edu/duplication/; Lee et al., 2013) and the visualization was carried out with CIRCOS (Krzywinski et al., 2009). The possible duplications between M. acuminata and A. thaliana as well as M. acuminata and O. sativa were also identified and visualized.
Identification of cis-Regulatory Elements
Custom Perl script was used to extract the 1.5 kb upstream regions from the translation start codon of the genes and was considered as proximal promoter sequences. To identify cis-regulatory elements, PLACE cis-regulatory element online server (http://www.dna.affrc.go.jp/PLACE/; Higo et al., 1999) was used.
In silico Expression Analysis
To identify the ripening related WRKY genes, expression levels in ethylene treated and untreated fruit transcriptome datasets of M. acuminata; dwarf Cavendish, genome AAA, var. Robusta, Harichhal (Asif et al., 2014b) was analyzed. Reads from both the datasets were mapped on the MaWRKY proteins using GSMapper (version 2.5.) and the TPM (transcript per million) and fold change for the annotated contigs were calculated. Ripening related expression of the identified WRKY genes was also analyzed using transcriptome datasets of M. acuminata fruit under acetylene response for 40, 60, and 90 days (D'Hont et al., 2012). In addition, to study expression of identified WRKY genes in response to biotic stress; root transcriptome datasets of M. acuminata under Fusarium oxysporum fsp cubense stress (Li et al., 2012) were used. The heat map for the annotated transcripts with their expression values were generated using MeV version 4.2 (Saeed et al., 2003).
To study the expression of the ortholog pairs of M. acuminata WRKY genes with the rice WRKY genes, the microarray cel files were downloaded for the rice development (GSE6893) abiotic stress (GSE6901) and heavy metal stress (GSE25206) condition. The microarray cel files were normalized using RMA in AffylmGUI package (Wettenhall et al., 2006) in R-bioC (ver 2.15) and normalized data was used for differential gene expression.
Plant Material and Treatments
Dessert (M. acuminata) and cooking (M. paradisiaca) varieties of banana were used to study gene expression during development and fruit ripening. M. paradisiaca is the hybrid of M. acuminata and M. balbisiana belonging to AAB genome. Fruits from three independent plants of each cultivar at different developmental stages were harvested to collect the samples. Banana fingers from the same whorl of the hand representing similar developmental stage were treated with 100 μL/L ethylene for 24 h at 22°C in dark and then allowed to ripen in air as described in Lohani et al. (2004). Other vegetative tissues were harvested from 6 month old banana plants growing in the field. For RNA isolation, samples were quickly frozen in liquid nitrogen before extraction or stored at −80°C for further use.
RNA Isolation and Expression Analysis
Total RNA was isolated from banana tissues according to previously described protocol (Asif et al., 2006). Each RNA sample was treated with DNase I Digest kit (Sigma-Aldrich, USA) to eliminate DNA contamination. The integrity and size distribution of total RNA was analyzed by agarose gel electrophoresis. A NanoQuant (Infinite® 200 PRO NanoQuant, Austria) was used for RNA quantification. DNA-free RNA (5 μg) was used for synthesis of first strand of cDNA by using Revert Aid First Strand cDNA synthesis Kit (Fermentas, USA) as per manufacturer's recommendations. The quantitative RT-PCR was carried out with an ABI 7700 Sequence Detector (Applied Biosystems, USA) using SYBR Green chemistry. The amount of cDNA was normalized by using amplification of housekeeping banana actin as an internal control. Differential expression of five M. acuminata WRKY genes (MaWRKY121, MaWRKY38, MaWRKY61, MaWRKY83, and MaWRKY119) during fruit ripening was studied. Details of the oligonucleotide primers used in the study are provided in Supplementary Table 1. These MaWRKY genes were subjected to RT-PCR to validate their response in various stages of M. acuminata (AAA genome; dessert variety) and M. paradisiaca (ABB genome; cooking variety) during their course of ripening. The data from real-time PCR amplification was analyzed in terms of comparative fold expression following 2−△△ct method. All the experiments were repeated using three biological replicates and the data were analyzed statistically (± Standard Deviation).
Results and Discussion
Identification of the WRKY Family Members
The WRKY domain sequences were downloaded from Plant Transcription Factor database (http://planttfdb.cbi.pku.edu.cn/; Jin et al., 2014) and used to construct the HMM profile using HMMER (version 2.3.2). This profile was used for the identification of WRKY gene family members from the protein sequence of M. acuminata and M. balbisiana. After removal of partial and redundant sequences, a total of 147 and 132 WRKY genes were identified from M. acuminata and M. balbisiana, respectively (Supplementary Table 2). These MaWRKY and MbWRKY genes were named on the basis of their location on their respective chromosomes (Supplementary Table 2). Interestingly, though the WRKY genes are present on all the chromosomes, higher abundance was observed on chromosome 7 and 4 in M. acuminata while only in chromosome 7 in M. balbisiana.
There have been three rounds of genome duplication in Musa sp after the divergence of Musa from Zingiberales (D'Hont et al., 2012). Though, these duplication events resulted in the expansion of many gene families in Musa, there have also been simultaneous loss of many genes resulting in the control of gene family members (D'Hont et al., 2012). Some of the footprints of the genome duplication events were also observed in the WRKY gene family members. Our analysis suggests presence of duplicons in WRKY genes family members throughout the genome. The two Musa genome duplication blocks span chromosome 1, 2, 4 and 5, 8, 11, respectively. A total of 118 genes were present as duplication pairs throughout the genome, some of the genes had more than one duplicons.
Classification of the WRKY Family Members
Based on the presence of the WRKY and Zinc finger domains, WRKY gene family members were classified into three groups. Group I WRKY gene family consists of two WRKY domains while Group II contains only one WRKY domain. Both these groups have same zinc finger motif pattern of C2-H2. Group III WRKY genes also contain one WRKY domain but with a different zinc finger motif pattern of C2-HC (Rushton et al., 1995; Eulgem et al., 2000). The M. acuminata and M. balbisiana WRKY genes were classified into these three groups on the basis on their similarity to A. thaliana (72) (http://www.arabidopsis.org/) and O. sativa (101) (http://systemsbiology.usm.edu/BrachyWRKY/WRKY/Rice.html) WRKY domains.
WRKY domains from 147 to 132 WRKY members from M. acuminata and M. balbisiana respectively were aligned with each other using multiple sequence alignment tool ClustalX2 (Supplementary Figures 1, 2). The identified WRKY members which contain two WRKY domains were named as "CTD" or "NTD" on the basis of presence of domain at the C- or N-termini of the protein respectively. This led to identification of 167 and 150 separate MaWRKY and MbWRKY domains, respectively.
Further, the conserved 60 amino acid region of the WRKY domains was extracted from A. thaliana, O. sativa, V. vinifera, and Z. mays WRKY genes. This led to development of dataset comprising 778 non-redundant WRKY domains including M. acuminata and M. balbisiana WRKY gene domains (Table 1). An unrooted ML phylogenetic tree was constructed using these WRKY domains (Figure 1). Based on sequence similarity, the WRKY domains could be classified into Group I, II, and III genes. The Group II was further classified in Group II a-e. In M. acuminata, 24 genes were categorized in Group I whereas 106 and 16 identified as members of Group II and Group III, respectively. In M. balbisiana, 20, 87, and 21 WRKY genes were categorized in Group I, Group II, and Group III, respectively (Table 1). There were few WRKY genes (3 in M. balbisiana and 2 in grape) which were not assigned to any group and these were named as NG (not grouped).
Figure 1. Unrooted phylogenetic tree of WRKY domains. The tree was constructed using WRKY domains from the complete WRKY gene families of Musa acuminate, Musa balbisiana, Arabidopsis thaliana, Oryza sativa, Zea mays, and Vitis vinifera. A total of 778 WRKY domains were used for the analysis. The unrooted phylogenetic tree was generated using Maximum Likelihood algorithm with 1000 bootstrap value using JTT algorithm in MEGA 5.2.
The Group II WRKY genes were further divided in five (a–e) subgroups. Analysis suggested that five MaWRKY genes (MaWRKY118, MaWRKY85, MaWRKY144, MaWRKY11, and MaWRKY99) were mis-annotated as Group IId in banana Genome hub on the basis of their annotation with A. thaliana. The phylogenetic analysis suggested that these WRKY genes are the members of the Group IIc. Therefore, the MaWRKY genes present in Group IIa, Group IIb, Group IIc, Group IId, and Group IIe contains 11, 21, 32, 25, and 17 members respectively. At the same time, 11, 16, 30, 14, and 16 genes were present in Group IIa, Group IIb, Group IIc, Group IId, and Group IIe, respectively in M. balbisiana. In Group I, all the C-terminal and the N-terminal WRKY domain were grouped independently (Figure 1).
WRKY genes formed monocot- and dicot-specific clades depicting that the WRKY genes have evolved simultaneously in both monocot and dicot plants from the common lineage (Figure 1). Interestingly, except for Group I NTD, phylogenetic analysis led to the formation of monocot-specific clades for all the groups. Group III had the largest monocot-specific clade comprising Musa-specific clades (Figure 1). There were few WRKY members which did not cluster in any of the three groups and were named Not Grouped (NG). Similar observation was also made in Setaria where such genes were included in Group IV (Muthamilarasan et al., 2015).
Within three groups of WRKY genes, there are four major lineages comprising Group IIa + Group IIb, Group IId + Group IIe, Group IIc + Group I (NTD and CTD), and Group III (Zhang and Wang, 2005; Rinerson et al., 2015). Till now, there were two proposed hypothesis regarding evolution of WRKY gene family in plants. According to Group I hypothesis, all the WRKY genes are evolved from the Group I-CTD (Zhang and Wang, 2005). In addition, alternative Group IIa+b hypothesis state that Group IIa and Group IIb evolved directly from the single domain ancestral algal WRKY genes separately from Group I WRKY genes (Rinerson et al., 2015). The phylogenetic analysis of the monocot and dicot plants favors the Group I hypothesis, stating that in the five plants selected, the evolution of the WRKY genes had occurred from the Group I ancestral WRKY genes (Figure 1).
Duplication and Ks Analysis
The gene duplication pairs for M. acuminata were downloaded from the PGDD (http://chibba.agtec.uga.edu/duplication/) database and the genes containing WRKY domains were filtered. There were 136 WRKY gene duplication pairs which corresponded to 118 genes. This suggests that some of the WRKY genes have more than one duplicated genes (Supplementary Table 3). This could be due to multiple rounds of whole genome duplication events which have occurred in the Musa genome. The Ks (substitution rate) of these gene pairs was plotted against the number of gene pairs. Analysis revealed presence of two peaks corresponding to α/β and γ duplication events (Figure 2). It is interesting to note that more duplication pairs were present from the γ duplication event which could have occurred earlier. The analysis also suggests that recent duplicates of WRKY genes might have been lost due to the genome rearrangements (Figure 2). Interestingly, mapping of duplication pairs on the chromosomes suggested that many duplication pairs correspond to block 4 and block 7 of the Musa duplication blocks as suggested by D'Hont et al. (2012).
Figure 2. Substitution rate of WRKY genes in Musa acuminate. The graph represents the ratio of synonymous (Ka) vs. non-synonymous (Ks) variations within the Musa acuminate WRKY gene family. X- and Y-axis represent the substitution rate and number of genes respectively. Green bars represent the substitution rate at the time α/β WGD and the red bar represents the substitution rate at the time of γ WGD.
The orthologs of WRKY genes from O. sativa were also identified from the PGDD database. A total of 101 ortholog pairs corresponding to 62 MaWRKY genes were identified. The presence of additional 42 WRKY genes in O. sativa suggests that some genes have more than one orthologs (Supplementary Table 3). Analysis also suggests presence of 112 homolog pairs of WRKY genes between M. acuminata and M. balbisiana. Out of these, 111 and 112 were from M. acuminata and M. balbisiana WRKY genes, respectively. This suggests that there are specific WRKY genes which might have transferred and were conserved during the course of evolution within the Musaceae family (Supplementary Table 3). It was also identified that the ortholog genes within a chromosome of M. acuminata follow similar pattern as the M. balbisiana genome, depicting the conserved nature in the WRKY gene pattern.
Gene Structure and Chromosome Localization
The gene structures of the identified WRKY genes were retrieved from the genomic sequence of M. acuminata and M. balbisiana. The coding sequences of all the identified WRKY genes were mapped on to their respective complete genomes to identify intron-exon boundaries. The gene structures of the WRKY genes showed group-specific intron-exon patterns similar to A. thaliana and O. sativa (Wu et al., 2005). The number of exons varied from 1 to 9 in most of the WRKY genes of both the Musa species. Maximum number of exons, 23 numbers, was identified in MaWRKY14 (Supplementary Table 4). In M. balbisiana, twelve genes contain more than nine introns and the numbers ranged from 11 to 25. It was also identified that a set of the WRKY genes in both the species contain an intron in between the WRKY domain as reported in A. thaliana and O. sativa (Wu et al., 2005).
It has already been reported that there are two types (R- and V-type) of introns which are present in between WRKY domain. R-type introns, the phase-2 introns, have a splice site directly on the R residue of the WRKY domain. On the other hand, V-type introns, phase-0 introns, have a splice site before the V residue, at the sixth amino acid after the second Cys residue, in the C2H2 zinc finger motif (Wu et al., 2005). In M. acuminata, 105 WRKY genes contain R-type intron while only 31 WRKY genes have V-type intron and 11 WRKY genes do not contain introns in between the WRKY domains. Similarly, in M. balbisiana, 88 and 25 WRKY genes contain R- and V-type introns respectively while 17 WRKY genes do not have any intron in between WRKY domain. Analysis suggested that V-type introns are present mostly in Group IIa and Group IIb while R-type introns are present mostly in all the other groups (Group I, IIc, IId, IIe, and III). WRKY genes which do not contain any intron in the WRKY domains, in both the genomes, are those WRKY genes in which domain are mostly present at the N-terminal of the group I. This loss of introns was considered as the result of intron turnover or due to reverse transcription of the mature mRNA followed by homologous recombination with intron-containing alleles (Wu et al., 2005; He et al., 2012). The positioning of the introns in between the WRKY domains was found in the ancestral WRKY genes. Thus, it can be hypothesized that the WRKY domains in the WRKY family member might have duplicated from the ancestral WRKY genes which contain introns, followed by the divergence rather than the formation of the similar genes through convergence events (Wu et al., 2005).
The genomic position of each identified WRKY gene in both the genomes was also used to identify the chromosome localization. Analysis suggests that WRKY genes are distributed throughout the genomes of the M. acuminata and M. balbisiana (Figure 3 and Supplementary Figure 3). It was found that the maximum number of the identified MaWRKY (22) are localized on to the chromosome 7 followed by chromosome 4 and 10 with 18 and 16 MaWRKY genes, respectively. Six MaWRKY genes are present in the uncharacterized region of the sequenced M. acuminata genome (Figure 3). In M. balbisiana, maximum number (18) of the WRKY genes is present on the chromosome 7 while minimum (6) on chromosome number 1 and 2 (Supplementary Figure 3). Ten MbWRKY genes were present in the uncharacterized region of the sequenced M. balbisiana genome. It was observed that MaWRKY75 and MaWRKY76 on chromosome 7 as well as MaWRKY120 and MaWRKY121 on chromosome 10 are clustered together as the difference between them is 30 and 3 kb, respectively (Figure 3). Analysis also suggests that the identified MaWRKY and MbWRKY genes are evenly distributed on the different chromosomes and the uncharacterized region of both the genomes in accordance with the size of each chromosome (Figure 3 and Supplementary Figure 3).
Figure 3. Physical map of MaWRKY genes showing their chromosomal locations. The genomic position of each identified WRKY gene was used to identify the chromosome localization on different chromosomes and the uncharacterized region. Vertical bars represent the chromosomes and numbers at the left indicate the position of genes (in Mb). The segmental duplicated genes are shown with red dots, and tandem duplicated with green lines.
Unique Motif Analysis
Ten conserved motif patterns were identified for both M. acuminata and M. balbisiana (Figure 4) WRKY genes. Of these 10 motifs in WRKY members from both the species, motifs 1, 2, 3, 4, 7, and 10 were identical in M. acuminata and M. balbisiana. Motif 6 of M. acuminata was identical to motif 8 of M. balbisiana (Figure 4). Motifs 5, 8, and 9 of M. acuminata and motifs 5, 6, and 9 of M. balbisiana were species specific.
Figure 4. Conserved motifs in WRKY proteins of Musa sp. The conserved motifs were identified in 147 MaWRKY and 132 MbWRKY proteins using MEME server with zoops models of minimum and maximum weight of 6 and 60 amino acid residues, respectively. The motifs that were present in both the Musa sp are represented in (A). Specific motifs that were present in only Musa acuminate are represented in (B) and those only in Musa balbisiana are represented in (C).
In M. acuminata and M. balbisiana, motif 1 and 2 are found to be conserved in all the identified WRKY genes while motifs 3 and 10 were conserved in Group I and few sub-groups of Group II WRKY members. Motif 4 was found to be kexin isozyme cleavage site while motif 10 is a pro-apoptotic protein. Motif sequence 1 is the basic WRKY domain of 20 amino acids which is found in all the WRKY genes. However, motif sequence pattern 2 is the variable sequence found in the zinc finger motif C2H2/C2CH (Figure 4). Motif 3 is the C2H2 conserve zinc finger motif which was found to be conserved at the N-terminal of the Group I WRKY genes (Figure 4). The Motif 6 of M. acuminata was similar to Motif 8 of M. balbisiana which is a fungi specific variant and was present in Groups IIa, IIb, IId, and III.
Some taxa-specific motifs were also identified between M. acuminata and M. balbisiana. In M. acuminata motif sequence 5 was found mostly in Group I and IIc while motif sequence 8 was present mostly in Group IIa and IIe. This motif is known to be responsible for the initiation of protein degradation. Motif 9 is phosphorylation recognition site present mostly in Group III, IId, and IIe (Figure 4). Likewise in M. balbisiana, motifs 5 and 6 are present on Group I, IIa, and IId while motif 5 is present in the Group IIb which is a phosphothreonine motif binding site. Motif 9 present in Group I, IIb, IIc, IIe, and III is a phosphorylation recognition site (Figure 4).
Apart from the basic 60 amino acid residue domain present in all the WRKY genes of different groups, there are differences in the WRKY domains present at the N and C terminal regions (Figure 4). These motifs are conserved in a particular group and thus might be involved in performing specific physiological and biological processes in plants. Therefore, it can be hypothesized that a specific group of WRKY genes might be responsible for the specific biological processes in plants (Kalde et al., 2003).
Analysis also revealed that the common motifs within M. acuminata and M. balbisiana WRKY members are the basic domain and the zinc finger motif. Also, the function of some of the motifs is similar in spite of the differences in their motif sequence pattern. Interestingly, Motif 9 present in both the Musa species have different sequence pattern but their function seems to be similar. This suggests that the function of the WRKY genes in a particular group is conserved and evolved simultaneously in specific taxa.
Apart from the basic WRKYGQK conserve motif in all the WRKY family members in M. acuminata and M. Balbisiana, WRKYGKK (in MaWRKY23, MaWRKY80, MaWRKY91, MaWRKY106, MbWRKY42, and MbWRKY92) and WRKYGRK (in MaWRKY37 and MbWRKY53) motifs were also identified. These variations in the WRKY domains are also reported in A. thaliana (Dong et al., 2003). Apart from these motifs, M. balbisiana WRKY members also contain WRKYGNK (MbWRKY59), WRKYGHK (MbWRKY54 and MbWRKY104), and WRKYGEK (MbWRKY122, MbWRKY123, and MbWRKY124) motifs (Supplementary Figure 1).
cis-Regulatory Elements
The 1.5 kb upstream regions of the WRKY genes were extracted and used for the identification of cis-regulatory elements using PLACE server and the cis-regulatory elements database. Many of the regulatory elements related to SA, cold, drought, ABA, and fungal response were identified in different genes. Interestingly, promoters from more than 50% of the WRKY genes harbor such elements indicating their important role during stress response (Figure 5). A set of WRKY genes also contain W-box and related motifs therefore regulation of these genes through other WRKY genes cannot be ruled out (Figure 5). A set of WRKY genes contain hormone-responsive elements of which majority of them are auxin and gibberellin response elements. A large number of genes also contain cytokinin, ethylene, and copper responsive elements (Figure 5). Previous reports have demonstrated that copper plays an important role in the signal transduction cascade of different hormones. A large number of genes also contain sulfur responsive element binding site which is known for essential for auxin dependent regulatory processes (Maruyama-Nakashita et al., 2005). Our analysis and previous studies (Knoth et al., 2007; Jiang and Deyholos, 2009; Levee et al., 2009) clearly suggest that WRKY genes play an important role during growth and development and stress responses. Interestingly, number of cis-regulatory elements identified in M. balbisiana is lesser as compared to those present in M. acuminate in the proximal promoter region used for the analysis (Figure 5).
Figure 5. cis-regulatory elements in WRKY genes of Musa sp. The cis-regulatory elements were identified in the 147 MaWRKY genes and 132 MbWRKY genes, respectively, using the PLACE server. Custom Perl script was used to extract the 1.5 kbp upstream regions from the translation start codon of the genes and was considered as proximal promoter sequences. Graph was plotted on the basis of presence of cis-regulatory element responsive to specific elicitors/conditions (x-axis) in WRKY gene family members (y-axis). The blue bars represent Musa acuminate WRKY genes and the red bar represents the Musa balbisiana WRKY genes.
The cis-regulatory elements identified, in this study, are evenly distributed throughout the promoter regions of the WRKY genes. The presence of SA, JA, ABA and ethylene related cis-regulatory elements suggest that these WRKY genes may be involved in various signaling pathways (Figure 5). The modulated expression of AtWRKY18 (Chen and Chen, 2002), AtWRKY70 (Knoth et al., 2007), and PtWRKY23 (Levee et al., 2009) has already been reported during fungi infection as well as in response to SA and JA. This suggests that a set of MaWRKY and MbWRKY genes might be involved in various defense related pathways. The other WRKY genes like GsWRKY20 (Luo et al., 2013), AtWRKY25, and AtWRKY33 (Jiang and Deyholos, 2009) GmWRKY13, GmWRKY21, and GmWRKY54 (Zhou et al., 2008) were also found to be induced during abiotic stresses. In M. acuminata, Group I and Group III WRKY genes contains regulatory elements responsive to salt, drought, and ABA stresses while Group II WRKY genes contain regulatory elements responsive to cold stress and pathogen infection. In M. balbisiana the same pattern was also observed apart from Group II genes which contained elements responsive to salt, drought, and ABA stresses (Figure 5).
In silico Expression Analysis
WRKY genes are known to be involved in growth and development as well as in stress responses (Maruyama-Nakashita et al., 2005; Jiang and Deyholos, 2009; Levee et al., 2009). In this study, involvement of WRKY genes in fruit ripening has been studied using the transcriptome data set of M. acuminata (available at the banana genome hub; (D'Hont et al., 2012)) as well as our in house transcriptome dataset of ripe and unripe fruit (Asif et al., 2014b). Thirty-one WRKY genes showed differential expression in the transcriptome data of D'Hont et al. (2012) of which 20 genes showed increase in expression during ripening with a fold change of ≥ 1.5 (Figure 6 and Supplementary Table 5). The WRKY genes were also mapped to the transcriptome dataset of ripe and unripe fruit (Asif et al., 2014b). A total of 64 genes mapped successfully in ripe and unripe fruit transcriptome of M. acuminata of which 14 genes showed enhanced expression (fold change ≥ 1.5) during fruit ripening (Figure 6 and Supplementary Table 5). Analysis using both the datasets suggests that a set of WRKY genes are differentially regulated during ripening process in M. acuminata. Analysis revealed that during ethylene induced fruit ripening, Group I MaWRKY genes are down-regulated while a set of Group II MaWRKY genes and most of the Group III MaWRKY genes are up-regulated (Figures 6A,B).
Figure 6. Expression of MaWRKY genes in response the ethylene, acetylene, and fusarium infection. To identify the ripening related WRKY genes, expression levels in ethylene treated and untreated fruit transcriptome datasets of banana (Asif et al., 2014a) was analyzed. Ripening related expression in response to acetylene exposure was analyzed using transcriptome datasets of M. acuminata fruit under acetylene response for 40, 60, and 90 days (D'Hont et al., 2012). Root transcriptome datasets under F. oxysporum stress (Li et al., 2012) were used to analyse biotic stress response of WRKY gene family. Log2 transformed count value are used for heat map construction. Panel (A) represents fold change between control (untreated) and ethylene treated (after 4 days of ethylene treatment. Panels (B–D) represent fold change between control (untreated) and acetylene treated after 40, 60, and 90 days, respectively. Panels (E), (G), (I) represent differential expression between control (untreated) and Foc1 treated after 3, 27, and 51 h, respectively. Panel (F), (H), and (J) represent differential expression between control (untreated) and Foc4 treated samples for 3, 27, and 51 h, respectively.)
To study the role of WRKY genes during biotic stress, WRKY gene expression was analyzed in transcriptome datasets related to F. oxysporum (Li et al., 2012) infection, available in public domain. It was observed that a large number (56) of WRKY genes expressed during F. oxysporum infected root tissue (Figure 6 and Supplementary Table 5). However, expression of WRKY genes was dependent on time duration post infection. There are 2 races (Foc1 and Foc4) of F. oxysporum which were studied at three time points (3, 27, and 51 h post infection; Figure 6 and Supplementary Table 5). While most of WRKY genes were up-regulated, decreased expression was observed for a set of WRKY genes post infection. Race specific differences in expression pattern were also observed as a set of WRKY genes expressed during early infection in Foc1 but not in Foc4 (Figure 6 and Supplementary Table 5). Expression of most of the WRKY genes continued to increase with time (Figure 6). During the pathogen attack in the M. acuminata plant, all the MaWRKY genes from Group II and Group III as well as some of the group I were up-regulated (Figure 6) suggesting response of specific banana genes during biotic stress response.
Since no public data is available to study the WRKY gene family expression during plant growth and development and other stresses in M. acuminata, the orthologs of the MaWRKY genes from O. sativa were retrieved and their expression was studied in available databases. Out of 147 WRKY genes in M. acuminate, only 62 had orthologs in rice database corresponding to 42 O. sativa WRKY genes (Supplementary Table 2). Analysis suggested that these WRKY genes are involved in the overall growth and development of the plant as well as in stress response (Supplementary Figure 4 and Supplementary Table 6). O. sativa WRKY genes were mostly up-regulated during abiotic stress (cold stress, drought stress, and salinity stress; Supplementary Figure 5A and Supplementary Table 6). In addition, data available in public domain (Chakrabarty et al., 2009; Dubey et al., 2010) was also used to study responsiveness of WRKY genes toward heavy metal stress. Analysis suggested that most of the WRKY genes are down-regulated during heavy metal stress (chromium, cadmium, arsenic, and lead; Supplementary Figure 5B and Supplementary Table 4). During the course of growth and development, Group I and III WRKY gene family members were up-regulated while Group II members were down-regulated (Supplementary Figure 4 and Supplementary Table 6).
In M. acuminata, most of the Group I WRKY genes were down-regulated during ripening and pathogen attack. Interestingly, their orthologos in O. sativa were up-regulated during abiotic stress and developmental processes (Figure 6, Supplementary Figures 4, 5). This suggests that the Group I WRKY genes were mainly involved in the developmental processes as well as in the abiotic stress response. Group IIc and Group IId genes are abundant in the set of WRKY genes regulating ethylene and acetylene related ripening. Our analysis suggests that in addition to involvement of Group II genes in fungal stress response, this group might also be responsible for ripening related gene regulation (Figure 6, Supplementary Figures 4, 5). Group III WRKY genes were found to be differentially regulated during early development of pollen and throughout the shoot development. Analysis also suggests Group III WRKY may not be involved in abiotic stress response (Figure 6, Supplementary Figures 4, 5).
The genes which showed differential expression under ethylene and acetylene exposure and fungal stress response had a high incidence of cis-regulatory elements related to elicitor response, ABREs and WRKY itself. This indicates that WRKY genes might be self-regulated by other WRKY members. Out of 147 WRKY genes, 26 genes contain the ethylene response element (ERELEE4). Out of these, 12 and 11 genes showed ethylene and acetylene responsive expression. Of these, five WRKY family members were commonly regulated by ethylene and acetylene.
Validation of MaWRKY Differential Expression
The differential expression of five M. acuminata WRKY (MaWRKY121, MaWRKY38, MaWRKY61, MaWRKY83, and MaWRKY119) genes was analyzed during fruit ripening using qRT-PCR. Our in silico analysis using different datasets of M. acuminata (dessert variety) revealed that these WRKY genes are differentially regulated during fruit ripening. RT-PCR analysis suggested that expression of MaWRKY121 is enhanced gradually during the banana fruit ripening process. In addition, MaWRKY83 and MaWRKY61 were late responsive as their expression enhanced significantly at the 6 and 8 day of ethylene induced fruit ripening respectively. No significant difference was observed in the expression of MaWRKY38 during course of fruit ripening. This is in contrast to the down regulation of MaWRKY38 gene in transcriptome analysis for ethylene and acetylene stress (Figures 6A,B).
Dessert (M. acuminata) and cooking (M. paradisiaca) varieties were also used to identify the possible role of banana WRKY genes in fruit development and ripening. These varieties have been used to study expression of members AP2/ERF and HDZIV gene families during fruit ripening (Lakhwani et al., 2015; Pandey et al., 2016). Expression analysis of five WRKY genes was carried out in different fruit developmental stages (3, 6, 15, 18, 21, and 24 weeks) and ripening stage of pulp (Figure 7). During fruit development, expression of MaWRKY38 gradually increased in the cooking variety and was highest at 21 week. Distinctly, in dessert variety, expression was significantly lesser and gradually decreased after 15 weeks (Figure 7A). The significantly higher expression of MaWRKY83 was observed at the 6 weeks in dessert variety (Figure 7A). This ripening related expression of MaWRKY83 is similar to as observed during ethylene induced ripening (Figure 7A). The expression of MaWRKY121 and MaWRKY61 did not vary much during the course of ripening in both dessert and cooking variety.
Figure 7. Expression analysis of selected WRKY genes during fruit development stages, ripening, and in different tissues. (A) Dessert (M. acuminata) and cooking (M. paradisiaca) varieties of banana were used to study gene expression during fruit development (in different weeks of development) and ripening (B) Expression analysis of WRKY genes in different tissues. Total RNA isolated from different tissues including Bract, Psudostem, Leaf as well as peel, and pulp tissues of ripe and unripe banana fruit was used for the analysis. The relative transcript abundance was normalized using banana actin gene.
The expression of selected MaWRKY genes was also studied at various tissues of the M. acuminata. It was observed that all the selected MaWRKY genes express mostly in stem as compared to other parts of the plant (Figure 7B). Highest expression of MaWRKY119 gene was observed in stem and leaf while MaWRKY83 gene was expressed mostly in leaf and unripe peel and was absent in unripe pulp (Figure 7B). Similar to WRKY gene family, genome-wide studies have been carried on few other gene families (Asif et al., 2014a; Lakhwani et al., 2015; Pandey et al., 2016) which has provided some inside about regulatory role of their members during fruit ripening and stress response.
In conclusion, this study suggests that WRKY gene family in M. acuminata and M. balbisiana is a large gene family. The gene family seems to have expanded before speciation, with minor changes in gene family after speciation. The expansion of the WRKY gene family was mainly due to the earlier γ duplication event of which most of the duplicons were lost in the recent α β duplication events. Our study suggests involvement of WRKY family members in various processes from development to ripening of fruit as well as biotic stress response in banana.
Author Contributions
MA and PT conceived and planned study. RG and AP carried out experiments. RG, AP, PT, and MA analyzed data. RG, MA, and PKT wrote the manuscript.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was supported by a research grant from the Council of Scientific and Industrial Research, New Delhi, as Network Project (BSC-107).
Supplementary Material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/article/10.3389/fpls.2016.00299
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Keywords: abiotic stress, banana, differential gene expression, fruit ripening, WRKY gene family
Citation: Goel R, Pandey A, Trivedi PK and Asif MH (2016) Genome-Wide Analysis of the Musa WRKY Gene Family: Evolution and Differential Expression during Development and Stress. Front. Plant Sci. 7:299. doi: 10.3389/fpls.2016.00299
Received: 31 December 2015; Accepted: 25 February 2016;
Published: 14 March 2016.
Edited by:Shabir Hussain Wani, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, India
Reviewed by:Abu Hena Mostafa Kamal, University of Texas at Arlington, USA
Mukesh Jain, Jawaharlal Nehru University, India
Copyright © 2016 Goel, Pandey, Trivedi and Asif. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Prabodh K. Trivedi, email@example.com; email@example.com;
Mehar H. Asif, firstname.lastname@example.com
†Present Address: Ashutoh Pandey, Department of Biotechnology, National Agri-Food Biotechnology Institute, Government of India, Mohali, India


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Who are
They're change agents who consistently put their community first, leading by example and serving others.
With that in mind, 80 Scholars, alumni, spouses and
The focus was on service and reenergizing and inspiring Coke Scholars to continue their commitment to positively impact their communities as everyday change agents. The weekend of inspiring speakers and projects to refresh Zavala Elementary School did just that.
"The idea is to have them come back here, be together, connect with each other, and remind themselves of the importance of service," said Jane Hale Hopkins, executive vice president,
Scholars ended the three-day conference by making service pledges that would impact their community once they return home. "We don't want to just serve the community, but we want to inspire others to also make a change," said Rima Patel, 2013
The 2016

subset: commoncrawl


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Fire Walk With Me
We live in a magical age people – I so love that a person is able to watch their favorite television shows on the internet. You don't have to wait for a specific time – you can pause them, watch them several times in a row – IT"S MAGIC I tellya MAGIC!
Lately, I have been obssessed with re-watching Twin Peaks. David Lynch is a freaking genius in my opinion. Even the stuff that you don't understand is fantastic. Watching this I was thinking how ahead of its time this show was. If you have some time – go watch it from the beginning.


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Thank You Donors and Volunteers! 233,408 Meals Served in 2014
Thank you donors and volunteers for an amazing year of service to the homeless, hurting and addicted in our community! In 2014, Union Gospel Mission provided 233,408 meals to the homeless and people in need.
2,544 food boxes were provided to individuals and families who were struggling just to keep a roof over their head. Because of you, those families were able to get a food box or even a special Thanksgiving basket with a turkey certificate and plenty of food for a wonderful Thanksgiving meal.
Thanks to your support we were able to serve 4,685 GALLONS of coffee to our homeless guests that enjoyed the hospitality of our day room. These guests were also provided with 124,208 snacks.
In addition to meals, food boxes and snacks, Union Gospel Mission provided 24,100 clothing items to the homeless and people in need. Including nearly 2,000 blankets and sleeping bags! This is possible because donors and volunteers like you!
Our guests also received 2,027 hygiene items, essentials like a toothbrush, deodorant or soap. In our day room and at our special events we were able to distribute 1,256 Bibles.
Beyond the essentials of food and clothing, the generous support of our donors and volunteers helped us celebrate our first year in our new LifeChange Center for Women and Their Children located in Beaverton. This new location and expanded program helped women and children escape abuse, homelessness, and addiction and find fresh hope in a safe, healing home! The new facility was filled with women discovering a path to healing from past traumas and bad choices, and children enjoying the safe environment to play, laugh and be children.
Our men's LifeChange program in our downtown location has been going strong through 2014. We are so thankful for donors and volunteers supporting men who were homeless and addicted find new purpose. Through pastoral counseling, the Genesis relapse prevention program, and work therapy men in LifeChange have been building new lives.
LifeChange for men, women and children is a Christ-centered program that is transforming lives in a powerful way!
None of this outreach and service would be possible without the over 3,523 volunteers that generously gave their time and talents to help! Thank you volunteers!
We are amazed and humbled by the support of our donors and volunteers in 2014. Thank you for blessing our neighbors in need and transforming lives through supporting Union Gospel Mission in 2014!
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Peace & Privacy! Located on Creamery Rd. in beautiful Monroe County. This 4BR, 1BA cinder block house has much to offer! Beautiful hardwood floors from 1955, french door, 12.55 acres (approx. 65% wooded / 35% open), pond, large fenced in garden spot, garage/workshop in basement, large rec. room in basement, outbuilding, etc. Home needs a little TLC & updating but has huge potential!
Call Robert Bittinger, Jr. 304-646-6411


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I thought you might like to have a nosy round Kate's house today. I used the pictures below to reference Kate's house in Blackfeather and as an aide memoire. It's very easy to forget exactly what colour a room is painted or where you left that gun, if you're writing a crime novel. Having a visual reference can help you get the details right from scene to scene and can save on editing at a later stage. Also, it's fun to be able to see where a character lives or works, the clothes they wear or the car they drive.
Strictly speaking this is Kate's parents house, but as they're now living in France they've all but given it to her.
And finally Kate's 1971 cherry red VW Beetle
Nel Ashley is the author of Blackfeather – a Fallen Angel Paranormal Romance and Immortal, the second book in the Blackfeather Series. She is currently working on her third novel, Persephone Reborn, a vampire romance influenced by Greek mythology.
You can also connect with Nel on Facebook and Twitter


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3 Dozen Mixed Gerberas In A Vase
This exudes style! Elegant white and yellowish gerberas are harmoniously arranged in a lovely and modern glass vase. Expertly designed with gorgeous ribbon decoration, this is a great gift to celebrate special occasions and to convey feelings. Do not hesitate to purchase it now.
Product Code: HCMTHRDZGER-921
Reward Points: 64
Availability: In Stock
Reward Points: 64
Availability: In Stock


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Here's your chance to do something TOTALLY INCREDIBLE for your office without any added effort…
And the best part about it is, it doesn't cost a lot and it won't monopolize your time.
You see, in today's hyper competitive world, finding good talent is very hard to come by… KEEPING good talent is even harder.
There always seems to be another company than can offer a little bit more, or have a better comp plan, or a better retirement plan that can steal your employees away.
Employees are overworked, stressed out, unhealthy, and costing their company millions in lost productivity and health care costs.
Top talent is hard to come by and even harder to keep, which is why the workplace environment and perks are so unbelievably important.
In a nutshell… SnackNation takes all the hassle off or your shoulders and just makes you look like the office Rock Star. Having SnackNation delivered to your office means:
Every month we'll send you a box stocked full of the best tasting and healthiest snacks out there. You'll also get a complimentary display box – It will look good in the office and help you keep everything organized.
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Select your subscription plan based on the number of employees you have in your workplace or the number of break rooms you want to stock.
Your first shipment will contain the SN display and 1 refill box. Designate someone to restock the display as additional refill boxes will arrive.
You'll hear a buzz around the office as employees try the tasty and healthy snacks. Their mid- afternoon energy crashes are replaced with smiles!
No worries because… The "Collection Box" solution was created for companies on a tight budget
Have you ever heard of an "honor box?" Employees grab a snack and then place a dollar in the attached collection box. The program is "on your honor" where they pay for the product they take. This is a system that has worked for years and years in the traditional vending industry.
The beauty of this program for your office is you have complete control over how much you charge for the product. You can make everything a dollar or fifty cents…it's up to you. If fact, with the Collection Box program, you can chose to have this box completely pay for itself every month.
For Example
An Easy, Hassle-Free Way To Get Top-Selling Delicious Healthy Snacks Delivered Right To Your Office


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It's Trinity Sunday again, one of my favourite Sundays of the year, because I like to think about the persons of God in relationship with each other, and with us.
When I was a teenager, I saw a whimsical short film called Jesus B.C., in which God was "Dad," Jesus was "Chris," and the Holy Spirit was a beautiful black woman referred to as "Grace." I wish I could find it and watch it again (I have tried, but can't track it down even in this digital age). "Dad" seemed to be the CEO of creation, and he had a hilarious archangel butler named Norman, who was having an interesting time reporting back to the boss as different Old Testament biblical stories were occurring (I can still see poor Norman standing under a little umbrella in the pouring rain as Noah's Ark floats past and lightning flashes). I can't even remember the point of the movie, but for me it made the very abstract idea of the Trinity more accessible through the conversations between Dad, Chris and Grace, their love for each other, and their fondness for the crazy humans who kept Norman busy reporting on their latest escapades.
Since then, the idea of God as a relational being has become a stronger and stronger theme in my life: God as tender parent, brother and sister, creator, lover, and inspiration for life. And the stronger this theme becomes, the harder it is to sit still in church -- because God is so much bigger than any one church or faith understands, and God's love is so much more inclusive than our tiny little hearts and minds can fathom, or our words and faith denomination can express. What I'm seeing is that each faith seems to have a number of rules and regulations, some of them designed by the "people in charge" to keep adherents in line. Unfortunately, those rules and regulations often end up excluding certain people, and when the excluded ones are treated unjustly by the elite (or elect), community breaks down. It's not hard to imagine Chris, Grace, and Dad shaking their heads.
Even though I'm a cradle Catholic (baptized when I was seven days old), I struggle with so many of my church's rules and regulations, and I know I'm not the only one. As a "good Catholic," I am supposed to be unquestioningly obedient to the Magisterium (a patriarchy-flavoured word designating the teaching authority of the Church) which has always scared the heebie-jeebies out of me because it sounds so... I dunno... so inhumane. But the older I get, the more I think that the Magisterium's insistence on unquestioning obedience is one of those things that prevents the true relationship and true community of the Trinity's embrace from taking hold of our hearts -- and our world. People expected to be unflinchingly loyal often aren't allowed to be honest about how they feel under that obedience, or to ask questions.
If the Catholic Magisterium wasn't quite such a misogynistic, power-hungry, male-dominated human institution stuck on enforcing some pretty archaic man-made rules, but rather, was a broad-minded community made up of men AND women of every race, creed, social strata, sexual orientation and state of life, it would be easier to follow. And if it would take Jesus' radical teachings to heart and live his Gospel of care for the poor and marginalized, I would jump on the bandwagon wholeheartedly. But at the moment, I'm standing firmly on the curb as the parade goes by because I have tons of questions that the Magisterium is choosing to ignore.
The most recent: on my birthday (Thursday) and his 15th anniversary as a priest, Father Gary Meier came out of the closet as a gay celibate servant of the people of God in the American Roman Catholic Church. He had previously published an anonymous book about being a gay priest, because he was afraid of the consequences of being honest. But now he has spoken out in compassion for other gay Catholics, priests and laity alike, and the Magisterium is suddenly paying attention in a very Magisterium-like way, focused on its rules about sexual ethics rather than on the honesty and bravery of one man.
And so, there are thousands of other priests, gay and straight, afraid to speak up, that aren't under the Magisterium's watchful eye. I know some of them, and love them, and honestly, their sexual orientation doesn't matter. They are good and faithful servants of the servants of God. So my question is, why is the Church, which insists it descends directly from Jesus, so hung up on sexual orientation and other such things? Jesus certainly wasn't. He was about setting people free!
The Trinity's watchful eyes focus on love, and on community. So what would the Trinity say about what Father Gary has done? I suspect they would rejoice that he has finally been able to speak the truth of who he is, and perhaps even celebrate the fact that he is taking a stand for Catholics who feel judged by the Magisterium because of their God-given sexuality. In fact, I can't imagine that the Trinity gives a hoot about whether any of us are gay or straight, Christian or Muslim, green or purple, as long as we are good and faithful.
In my younger years, I'll admit that I felt uncomfortable with the idea of homosexuality, simply because the gay people I knew couldn't be open about it so that I could learn that it was different, but okay. When I was growing up, gays were mistreated and made fun of, so how could anyone be open or honest? However, as society began to acknowledge that heterosexuals weren't the only authentic lovers on the block, things changed. When some of my friends came out of the closet, they challenged my archaic Catholic understanding of homosexuality as Sodom and Gomorrah's evil (I now wonder if those legendary places weren't examples of other sorts of iniquity like slavery, genocide, racism, and unbridled capitalism). My gay friends are good people who have converted me, making me see that sexual orientation is like all those other things that make people different from one another -- differences God created to enrich humanity.
God created the world and each of us, and if we believe what the book of Genesis tells us, God calls everything good, embracing everything and everyone in a love that's beyond our understanding. Grace, Dad, and Chris just want us to love, to be happy, to know the goodness of the Trinity, and not to judge or exclude each other because of our differences.
So if we want to grow in the image of the Trinity, in love and community, we need to embrace each other's differences, too.
Thank you, Holy Trinity,
for molding creation in your image and likeness.
What an incredible being You are,
full of diversity -- and unity.
Open our minds and hearts to the many ways You are manifest in all those around us.
Help us to see You in everyone we meet,
and to welcome them as You will one day welcome us all.
Simple Moodlings \'sim-pѳl 'mϋd-ѳl-ings\ n: 1. modest meanderings of the mind about living simply and with less ecological impact; 2. "long, inefficient, happy idling, dawdling and puttering" (Brenda Ueland) of the written kind; 3. spiritual odds and ends inspired by life, scripture, and the thoughts of others
Sunday, May 26, 2013
Trinity Sunday: God's embrace... and ours
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About AGL Resources
AGL Resources (NYSE: GAS) is an Atlanta-based energy services holding company with operations in natural gas distribution, retail operations, wholesale services, midstream operations and cargo shipping. As the nation's largest natural gas-only distributor based on customer count, AGL Resources serves approximately 4.5 million utility customers through its regulated distribution subsidiaries in seven states. The company also serves more than one million retail customers through its SouthStar Energy Services joint venture and Nicor National, which market natural gas and related home services. Other non-utility businesses include asset management for natural gas wholesale customers through Sequent Energy Management, ownership and operation of natural gas storage facilities, and ownership of Tropical Shipping, one of the largest containerized cargo carriers serving the Bahamas and Caribbean region. For more information, visit www.aglresources.com.


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Nevada residents who have suffered an injury because of the actions or inactions of another person may wonder if they have a personal injury case on their hands. Laub and Laub, attorneys at law, define what can be filed under the broad category of personal injury claims.
Many different potential incidents can fall under personal injury claims, as it's a very large umbrella term. A number of incidents are related to motor vehicle accidents or other road-related crashes. This can include accidents on or in:
- Motorcycles
- Trucks
- Cars
- Bicycles
- Pedestrian
Fatalities or wrongful death resulting from injury will still fall under this category, even though the victim passed. Both catastrophic and serious injuries can apply as well. This can include burns, electrocutions, loss of limbs, and severe trouble with organs.
Personal injury claims exist to allow you to reap the compensation that you're owed for your injury. This compensation will help you to cover the cost of medical bills, medication, surgery, time off, and any other financial cost that may come to you due to your injury. If you do not try to obtain compensation, you may be left trying to pay for these things on your own, adding financial stress to your emotional burden.
Dealing with personal injury cases can seem complex. However, if the negligent party is held responsible, it can make life much easier for you after an accident. For this reason, you may wish to consider filing a case. You may benefit from taking a look around our web page for more information.


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Hi everyone. Hope you are all doing well. I was so excited to come home to my first set of ATC cards send from Marions ATC club. I can't believe the work out there.
I haven't been doing any crafts lately. I've lost my mojo. I just can't seem find the time or inspiration to do any projects. I want to make christmas cards and haven't started yet. & I also need to scrapbook. My daughter has changed so much since the summer, I've taken so many pictures but have done nothing with them yet. And I promise myself to do it soon.
Anyway, that is all for now. Chat with you again soon.
Oh ya, I'll be out of town this weekend,but I'll check in once I get back.
Tuesday, October 21, 2008
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By Hadi Hafidz (Photos: Public Affairs Department)
Navy blue has been a shade synonymous with our police uniform. Since April 2018, a batch of officers have been seen sporting a brighter shade of blue. Attired in bright blue polo tee shirts, black cargo pants and black sports shoes, these are our Volunteer Special Constabulary (VSC) (Community) officers who have recently joined the Singapore Police Force (SPF) to prevent, deter and detect crime!
Specially tailored to suit those who are keen to volunteer but are concerned about not being able to commit the time, the VSC (Community) vocation was created to provide more opportunities for the public to volunteer with the SPF. This new vocation now only requires volunteers to contribute eight hours per month and has a shorter training duration that is conducted over seven weeks. VSC (Community) officers will be trained in law, Police procedures, Police Defence Tactics (PDT) and first aid.
Upon completing their training, these officers will be deployed at the Neighbourhood Police Centres (NPCs) to conduct foot patrols in community areas with high human traffic.
In this issue of Police Life, we caught up with some of our VSC (Community) officers who let us in on their motivations and their experiences thus far.
Special Constable (V) Amy Cheow Poh Yen,
Clementi Neighbourhood Police Centre (NPC)
42 years old, Field Application Support Engineer, Rudolph Technologies Incorporation
What inspired you to join as a VSC (Community) officer?
I was introduced to the SPF's volunteer schemes by two Community Policing Unit (CPU) officers who visited my home to share about the SGSecure movement. The sharing session brought back memories of a terror attack incident that took place when I was in New York City. A lorry had ploughed into the bikeway and killed eight people. The thought of that incident prompted me to sign up for the vocation as this is an opportunity for me to contribute to the safety and security of our home.
Which part of the training do you feel is most important?
Through the first-aid lessons, I have learnt very useful skills such as Cardiopulmonary Resuscitation (CPR) and various bandaging techniques. Such skills could make a difference in situations of life and death!
What was your most memorable experience while on patrol?
I remembered an incident when we were approached by a drunkard during our foot patrol. When we asked him for more information, he started getting agitated. Together with the CPU officers who were patrolling with us, we managed to calm him down. I learnt that policing was not just about the hard skills but also the soft skills that are equally important in helping us to engage the community effectively.
Special Constable (V) Khanna Moneesh Kishanlal
Choa Chu Kang NPC
47 years old, Vice President, Oversea-Chinese Banking Corporation Limited
My interest was piqued when the CPU officers shared about the volunteer schemes in the SPF during one of their house visits. As they were sharing, I thought about the time when my family and I migrated to Singapore 17 years ago and how the community helped us to integrate into society. The peace and harmony that we have enjoyed over the years cannot be taken for granted. So I decided to take this opportunity to do my part in keeping Singapore safe.
Which part of the training do you feel is most important?
As a newbie to policing work, I found the PDT training very useful as such skills would help me to carry out my duties confidently.
What was your most memorable experience while on patrol?
During one of my patrol duties at a heartland mall, we were informed by members of the public that a man had sustained a fall. When we reached the incident location, we learnt from the man that he had lost his balance and fell. After assessing that he had no injuries, we helped him to rest at a nearby restaurant. We chatted for a while until he felt stable enough to walk. He expressed his gratitude and we parted ways. I was happy not only because I had helped someone, but also because the residents appreciated our presence and turned to us for assistance.
Special Constable (V) Muhammad Imran
24 years old, Undergraduate at National University of Singapore
What inspired you to join as a VSC (Community) officer?
It all started when I received an email, sent to all recallable Police Operationally-Ready National Servicemen, about volunteering opportunities with the SPF. As I learnt more about the vocation, I was intrigued by the idea of how I could contribute to crime fighting efforts through engaging the community and at the same time, juggling with my study commitments. When I was serving my National Service with the SPF, I was deployed as a Police Coast Guard officer, spending most of my time patrolling the Singapore waters. As a VSC (Community) officer, my policing terrain would be significantly different. Hence, this piqued my interest to join.
Which part of the training do you feel is most important?
The knowledge gained from the law lessons were very useful as it would help to guide us on the necessary actions to take when we come across incidents. The trainers also made the lessons interesting by including some real life examples.
More than 50 VSC (Community) officers have since been appointed and deployed for duties, with the numbers steadily increasing. The fourth batch of officers, including Special Constable (V) Imran, underwent their training and graduated on 30 October. In our bid to expand the VSC (Community) family, the SPF will continue our efforts to encourage like-minded individuals to volunteer with us. If you know of friends or family members who are keen to contribute to the safety and security of our community, do encourage them to drop us an email at email@example.com or scan the QR code and register with us!
Keen to contribute even more?
The VSC scheme is available for those who can contribute a minimum of 16 hours of duty per month. Volunteers will be equipped with firearms after undergoing six months non-residential training inclusive of basic police skills, basic legal knowledge, street craft, firearms training, physical training, drills and unarmed combat.


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Why North Carolina's loss to Duke in the ACC tournament is not necessarily a bad thing
UNC's final shots did not find the basket in the loss to Duke
North Carolina had to give credit where credit was due.
In part three this season in the UNC-Duke rivalry, Duke freshman Zion Williamson, who missed all but 36 seconds of the first two games after spraining his knee, was unstoppable Friday in the semifinals of the ACC tournament.
He scored 31 points, including the game-winning put-back layup with 31 seconds left. The offensive rebound came off Williamson's own miss, but he was too quick and jumped higher than the rest of North Carolina's players to corral the rebound and score.
"The guy that's been hurt came back and put on his superman jersey again and was incredible," North Carolina coach Roy Williams said.
The Tar Heels led Duke by 13 points with a little more than six minutes left in the first half. They appeared to be the better team and looked more confident. Williamson started 2 of 6 from the floor. But he made 11 of his last 13 shots to help the Blue Devils grab momentum and win 74-73.
"He's a good player, he's explosive, he's very talented," UNC senior Luke Maye said of Williamson. "But I mean, it comes down to a team game, I thought it was a great game and we didn't have the ball bounce our way."
The Tar Heels had multiple cold stretches in the second half. After taking a 65-63 lead with 8:22 left in the game, UNC did not make another field goal for nearly five minutes.
Duke coach Mike Krzyzewski put Duke reserve guard Jordan Goldwire on UNC graduate Cam Johnson, who had 16 points in the first half. The move worked. Goldwire had two steals in 28 minutes. He held Johnson to 3-of-10 shooting in the second half.
The Tar Heels had trouble scoring after Duke finally gained momentum. Their offense became stagnant, and the shots they took were often contested.
UNC shot 40 percent from the floor as a team, and finished 4-of-27 from the 3-point line. Of the five Tar Heels who took more than three shots, none shot above 39 percent. The Tar Heels struggled from behind the 3-point line in the first game against Duke, too, finishing 2 of 20. But the difference in that game, was they dominated in the paint.
In this game, Duke outscored UNC in the paint 50-38. Credit Williamson, who had 22 points in the paint.
"We know we've got to get better on offense, that's pretty clear," UNC senior Kenny Williams said.
The Tar Heels' loss to Duke snapped an eight-game winning streak that included the two wins over Duke without Williamson. The Tar Heels were also eliminated from the ACC tournament, as Duke advances to the finals to face No. 4 seed Florida State.
"There's no relief when you lose a game," Johnson said. "And this was one that we wanted to win, this is one that we thought we should win, this is one we could have won."
Said, Maye: "It's tough to go out like that. We've just got to get better."
The loss for the Tar Heels doesn't mean the end. They lost to Duke, a top five team in the polls, by one point with three shots at the rim to win it. It was also only their second loss in their last 16 games. And the winner of the ACC tournament does not always win an NCAA title.
Of the 13 times ACC teams won an NCAA title in the last 50 years, only six won the ACC tournament that year. When North Carolina won it all in 2017, the Tar Heels lost to Duke in the semifinals of the ACC tournament. The Tar Heels did not win the ACC tournament in 2005 or 2009, when they won national titles.
"We'll learn from our mistakes and those we made in this game, and we'll use that to propel us to a national championship," UNC junior wing Brandon Robinson said. "It's not the end of the world. You can win six games in the NCAA tournament and it will change your whole life."


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What's new
London South Bank University recently won the "Accountancy College of the Year" in the Public Sector category for the second year in a row at the PQ Magazine Awards.
The University was one among the many entrants for this year's competition in the category against such institutions like Belfast Metropolitan College, CIPFA, University of Derby and Gower College Swansea.
Mike Molan, Dean of the School of Business, picked up the award on 20 February. He said, "I am extremely proud of the team here at LSBU for their hard work and dedication to our students – we continuously aim to deliver excellent student satisfaction, from offering relevant and high quality teaching, to numerous extracurricular activities and opportunities for our students to take part in."
LSBU alumnus, Tyson Nsimbe, won Part Qualified accountant of the Year. He is currently pursuing a career as an Accountant at FairFX PLC, following his degree in Accounting and Finance.
"Tyson embodies everything that LSBU stands for and is definitely deserving of the award," said Sarah Moore-Williams, Director of Education and Student Experiences at LSBU. "I have known Tyson from his first days at LSBU and he has continued to impress me but also constantly surprises me for his devotion to his career and willingness to give back to LSBU."


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Security is a looming issue for organizations. The threat landscape is increasing, and attacks are becoming more sophisticated. Emerging technologies like IoT, mobility, and hybrid IT environments now open new organization opportunity, but they also introduce new risk. Protecting servers at the software level is no longer enough. Organizations need to reach down into the physical system level to stay ahead of threats. With today's increasing regulatory landscape, compliance is more critical for both increasing security and reducing the cost of compliance failures. With these pieces being so critical, it is important to bring new levels of hardware protection and drive security all the way down to the supply chain level. Hewlett Packard Enterprise (HPE) has a strategy to deliver this through its unique server firmware protection, detection, and recovery capabilities, as well as its HPE Security Assurance.
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iPhone & Other 'Smart Phone' Photography
Next Monday, 27th February we will be looking at the increasing use of mobile phones, tablets and other similar electronic devices for the taking, viewing, sharing, and nowadays even the processing of photographs. The session will be led by Club members Ray Portman, Nigel Reader, Peter Rean and Duncan Turner, but the aim is for this to be an interactive evening with members contributing their own experiences and views on this popular photographic activity.
To show what can be achieved, we invite members to submit a maximum of two images taken with a mobile phone, iPad, GoPro, or similar device. From the entries submitted Ray, Nigel, Peter & Duncan will select one to receive a top quality memory card as a prize.
Images can be submitted in any size and format, but preferably as JPEG's and you should provide your own name and a title for the photograph. All images will be put into a slideshow and displayed on the large screen on the evening. Get your entries in by midnight on Wednesday 22nd February by sending them to Ray at email@example.com


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Located in the South of Aberdeen, our client is looking to appoint a Tax Advisor on a permanent basis.
Duties include:
The ideal candidate will have a degree in accounting and will be either part-qualified and or fully qualified.
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text: Leading Indicators: What Are They & How Can They Improve Your Business? February 22nd, 2018 Beth Perry Beth Perry Beth Perry Read More About Beth Beth Perry As retail becomes increasingly competitive, commerce marketers – particularly those in the enterprise space – are under constant pressure to come up with new ways to generate revenue. Many are looking to big data, which is promising – but it can also present problems. Whenever a consumer interacts with your brand, a numerical footprint is created. When thousands of shoppers are creating those footprints at various stages of the funnel, it can be easy to become overwhelmed by the flood of metrics: click-to-open rates, conversions, number of email sign-ups, cart sizes. And it hardly stops there. Fortunately, you can use a number of strategies and tactics to sift through these metrics. Our latest white paper, in collaboration with Shopify Plus, Leading Indicators: KPIs for Ecommerce and Tactics to Move the Needle, outlines a few of them and reviews how to determine which ones are most valuable for your brand. Focus on Leading Indicators Identifying the data with the biggest influence on your brand and revenue can certainly be challenging. Which metrics really matter? Where should you apply your energy? How can you find your focus? It all depends on your business goals or key performance indicators (KPIs). KPIs are either leading or lagging, and because they're easier to measure, many businesses tend to focus on lagging indicators, such as revenue, profit and costs, to track performance. But these companies are missing out: If you're not concentrating on leading indicators, you risk losing your edge as the market continues to evolve. Leading indicators are imperative to your brand's success. By measuring them, you can predict possible future outcomes and make adjustments to save time, resources and revenue. Let the Data Direct Your Strategy Deciding what leading indicators matter for your company is essential. To find the answer, you'll need to test out different tactics and track what's working – and what's not. As you fine-tune your strategies, adjust as needed based on the data you're receiving. Identify the key metrics that matter for your brand, create and implement a plan, then start measuring and tweaking as you go – and remember to maintain consistency in your reporting. Your shoppers are constantly providing you with the data you need to convert them into loyal and satisfied customers, but it's up to you to skillfully apply it to your business strategy and create a unified, creative and effective modern retail experience. The mystery of converting customers can become a straightforward map – and you get to decide its coordinates. You decide how to navigate it – and the possibilities are endless. To learn more, download Leading Indicators: KPIs for Ecommerce and Tactics to Move the Needle. Read More on This Topic: Measuring Matters: Defining, Benchmarking, Evaluating and Improving Your Email Campaigns Marketing Personalization: Let the Data Do the Work Leading Indicators: KPIs for Ecommerce and Tactics to Move the Needle


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Makes Me Happy/
I have a random (rather large) painting of a dog in my den/library/room-we-don't-know-what-to-call...... She's a little wonky.... One leg doesn't seem attached in the right place exactly. Her perspective isn't quite right. But I love her. She was a gift from my brazen daughter-in-law who talked the antique/junk store owner down to $15. Sometimes when new friends come to the house they will ask about her, if she is a beloved pet, etc. I like to make up silly stories about her like she was my grandmother's dog who dug granny out of a snowbank after an avalanche thus saving her life as a child and ensuring my future existence. Eventually I do tell the truth - she's just a random painting that makes me happy. Some people get it and some don't. I have a giraffe painting that a friend gave me that is kind of the same. He was taken down when we did some painting and redoing a couple of years ago. I think he's in the attic now. I'll have to get him out and find a home for him again. Hubs will be thrilled! :-P