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Maize (Zea mays) endosperm is a primary tissue for nutrient storage and is highly differentiated during development. However, the regulatory networks of endosperm development and nutrient metabolism remain largely unknown. Maize opaque11 (o11) is a classic seed mutant with a small and opaque endosperm showing decreased starch and protein accumulation. We cloned O11 and found that it encodes an endosperm-specific bHLH transcription factor (TF). Loss of function of O11 significantly affected transcription of carbohydrate/amino acid metabolism and stress response genes. Genome-wide binding site analysis revealed 9885 O11 binding sites distributed over 6033 genes. Using chromatin immunoprecipitation sequencing (ChIP-seq) coupled with RNA sequencing (RNA-seq) assays, we identified 259 O11-modulated target genes. O11 was found to directly regulate key TFs in endosperm development (NKD2 and ZmDOF3) and nutrient metabolism (O2 and PBF). Moreover, O11 directly regulates cyPPDKs and multiple carbohydrate metabolic enzymes. O11 is an activator of ZmYoda, suggesting its regulatory function through the MAPK pathway in endosperm development. Many stress-response genes are also direct targets of O11. In addition, 11 O11-interacting proteins were identified, including ZmIce1, which coregulates stress response targets and ZmYoda with O11. Therefore, this study reveals an endosperm regulatory network centered around O11, which coordinates endosperm development, metabolism and stress responses.
Maize (Zea mays) is one of the most important crops in the world and a model system for genetic and molecular biology studies. Maize endosperm, as a primary storage tissue, is a product of double fertilization, which produces a diploid embryo as well as the triploid endosperm (Russell, 1992; Kiesselbach, 1949). The fully developed maize endosperm includes four highly differentiated cell types: the aleurone (AL), the starchy endosperm (SE), the basal endosperm transfer layer (BETL), and the embryo-surrounding region (ESR) (Olsen, 2001; Becraft and Gutierrez-Marcos, 2012; Leroux et al., 2014). The AL is a single peripheral layer of cells that produces hydrolytic enzymes to mobilize storage in the SE when the seed begins germination. The SE stores starch and storage protein for the preparation of seed germination. The BETL is a dendritic transfer cell layer that transports nutrients from the maternal tissue to the endosperm. The ESR is defined by a group of cells that share ESR gene family expression and is believed to act as a physical barrier and a messenger between the endosperm and the embryo (Opsahl-Ferstad et al., 1997).
Maize endosperm development is controlled by sophisticated regulatory networks, with transcription factors (TFs) playing important roles. Genetic and molecular evidence has revealed a number of TFs that have key functions in endosperm development. Naked endosperm 1 (NKD1) and NKD2 are duplicate indeterminate domain TFs that regulate endosperm cell patterning and differentiation (Yi et al., 2015). Gontarek et al. (2016) demonstrated that NKDs modulate a series of biological processes in endosperm development, including gene expression, epigenetic functions, cell growth and division, hormone signaling, and resource reserve deposition and are direct upstream regulators of Opaque2 (O2) and Viviparous1 (VP1). VP1 functions as an activator of abscisic acid-induced genes for seed maturation and is also a repressor of germination-specific genes (McCarty et al., 1991; Hoecker et al., 1995). Another maize endosperm-specific DOF TF, ZmDOF3, is reportedly a direct activator of NKDs and a key regulator in starch accumulation and endosperm development (Qi et al., 2017). Thus, the identification and functional analysis of key TFs are important for understanding the mechanisms of endosperm development.
Starch and storage proteins are the primary nutrient components of maize endosperm, with starch accounting for ∼75% of the dry weight of maize kernels. Starch is synthesized in specialized amyloplasts via a process that involves the activity of four major enzymes: granule-bound starch synthase, starch synthases, starch-branching enzymes, and debranching enzymes (James et al., 2003). Protein accounts for ∼8% of the maize kernel dry weight, with ∼60% of this protein being prolamins, also named zeins. Cytoplasmic pyruvate orthophosphate dikinase 1 and 2 (cyPPDKs) are considered key molecular switches in the balance between starch and protein accumulation (Sheen, 1991; Prioul et al., 2008; Manicacci et al., 2009), and as an endosperm-specific bZIP TF, O2 positively regulates the cyPpdk genes and nearly all zein genes (Schmidt et al., 1987; Li et al., 2015). Loss of function of O2 results in a drastically decreased zein content and smaller protein bodies (PBs). Prolamin-box binding factor (PBF), another endosperm-specific DOF TF, directly interacts with O2 to regulate zein synthesis (Vicente-Carbajosa et al., 1997; Wu and Messing, 2012) and cyPpdk genes (Zhang et al., 2016). Therefore, elaborate regulatory networks involving TFs ensure efficient nutrient storage in the maize endosperm, though only a few have been studied to date.
In this study, we report the identification of a classic maize mutant, opaque11 (o11) (Nelson, 1981). o11 exhibits a reduced endosperm and unbalanced starch and protein accumulation. Through positional cloning, we found that O11 encodes a cereal endosperm-specific basic helix-loop-helix (bHLH) TF. Genome-wide identification of its binding targets revealed a regulatory network centered around O11, which coordinates cell development, storage nutrient metabolism, and stress responses in the maize endosperm.
o11 Has a Reduced and Opaque Endosperm with Developmental Defects
The o11 mutant obtained from the Maize Genetics Cooperation Stock Center was crossed into the W22 genetic background. On the F2 ears, o11 kernels exhibited 1:3 segregation to the wild-type kernels, indicating that o11 is a single recessive mutant (Supplemental Figure 1A). Mature o11 kernels had less vitreous endosperm and were significantly smaller compared with wild-type kernels (Figures 1A to 1C). The 100-kernel weight of o11 mutants was 32.3% less than that of the wild type (Supplemental Figure 1B). In the o11 mutant, the endosperm was more severely affected than the embryo by the mutation of O11 (Figure 1D; Supplemental Figures 1C and 1D). The SE region was greatly reduced, and its central region was empty (Figures 1D and 1E). The morphology of the o11 embryo was also affected in that the middle region of the o11 embryo's scutellum was thicker and the bottom region was narrower compared with the wild type (Figure 1E). Furthermore, we observed morphological changes around the ESR: Although an apparent interspace between the embryo and the ESR region was found in wild-type kernels, little space was observed in the same region in o11 mutant kernels (Figure 1F; Supplemental Figure 2). Wild-type kernels also exhibited clear and smooth boundaries at the interface between the scutellum and the surrounding endosperm, whereas o11 showed unclear and zigzagged boundaries (Figure 1F; Supplemental Figure 2). The germination rate and reproductive growth of homozygous o11 mutants were not affected, despite a mild growth delay in early seedlings.
o11 Has Less Starch and Protein Accumulation
Cytological observation of both the developing (Figure 2A) and mature (Figure 2B) endosperm indicated that the starch granules in o11 endosperm were smaller than those of the wild type. PBs were also smaller in o11 than in the wild type (Figure 2C). Biochemical analysis indicated that compared with the wild type, the total starch, protein and lipid contents of o11 endosperm per kernel were all decreased (∼34.2% less for total starch, ∼25.0% less for total protein, and ∼15.3% less for total lipid) (Figures 2D to 2F). When these storage nutrients were measured according to equal biomass, we found the starch content of the o11 endosperm to still be decreased per weight (Supplemental Figure 3A), but the total protein and lipid contents were increased (Supplemental Figures 3B and 3C). The C/N ratio of the o11 endosperm was also decreased compared with that of the wild type (Figure 2G). Moreover, the o11 endosperm showed a higher total amino acid content for most amino acids (Supplemental Figure 4A), and the free proline content of o11 was four times higher than that of the wild type (Supplemental Figure 4B).
Map-Based Cloning and Identification of O11
Using 9900 mutant kernels from a BC1F1 population, the O11 gene was genetically mapped to a 900-kb genomic interval on chromosome 2 (Figure 3A). This interval contained 14 predicted genes. RNA-seq analysis of wild-type and o11 kernels at 15 d after pollination (DAP) revealed only a few single-nucleotide polymorphisms in most of the predicted genes. However, a 3864-bp retrotransposon insertion was found at the 3ʹ end of the 2nd exon of GRMZM2G147685 (Figures 3B to 3D).
A functional complementation test was performed by transforming an 8464-bp GRMZM2G147685 genomic fragment amplified from the wild-type genome (containing 2311 bp upstream of the start codon and 1019 bp downstream of the stop codon) into maize (pApB, Hi-II hybrid). Based on genotyping analysis, o11/o11 kernels carrying the transgenic GRMZM2G147685 genomic fragment were functionally complemented, with the o11 mutant pheotype rescued (Figures 4A to 4C). We also produced GRMZM2G147685 knockout mutant alleles (five alleles, o11-cas9-1 to o11-cas9-5; Supplemental Figure 5A) using the CRISPR-Cas9 system (Qi et al., 2016a) and performed allelism tests by crossing two independent knockout alleles (o11-cas9-1 and o11-cas9-2) to o11-ref (Figures 4D to 4G; Supplemental Figure 5B). In allelism tests, kernels displayed a mutant phenotype similar to that of o11, indicating that o11-cas9 alleles cannot complement o11-ref (Figures 4E and 4F; Supplemental Figure 5B). All these results indicate that GRMZM2G147685 is O11.
Using an antibody raised against the protein encoded by GRMZM2G147685, we detected a single specific band with a molecular mass of ∼90 kD in wild-type kernels, whereas this protein was not detectable in o11-ref or o11-cas9-1 kernels (Figure 3E). Therefore, the loss of O11 function is caused by a retrotransposon insertion in the GRMZM2G147685 coding region, which results in the absence of the O11 protein.
O11 Encodes a bHLH TF Specifically Expressed in the Endosperm
O11 is predicted to be a bHLH protein without other known conserved domains; the full-length protein contains 624 amino acids, with the bHLH domain from residues 379 to 430. Phylogenetic analysis showed that O11 homologous proteins could be grouped into two major clades represented by monocotyledonous and dicotyledonous species, respectively. O11 grouped into the monocotyledon clade, whereas ZHOUPI (AtZOU) from Arabidopsis thaliana (Yang et al., 2008) was in the dicotyledon clade (Figure 5A; Supplemental Data Set 1). Notably, O11 contained an extra 300 amino acids at the N terminus, which was found only in Sorghum bicolor and Setaria italica (Supplemental Figure 6).
O11 transcript accumulation was observed only in kernels, with a peak at 9 DAP (Figure 5B). At the protein level, O11 was detectable only in the endosperm (Figure 5C). O11 was first detectable at 9 DAP, peaking at 18 DAP and decreasing thereafter (Figure 5C). Immunofluorescence assays with o11 mutant kernels as the negative control indicated that O11 was preferentially located in the nuclei of SE and ESR cells (Figure 6A). The subcellular localization of 35Spro:O11-YFP expressed in onion epidermal cells was mainly in the nucleus, with some localization in the cytoplasm (Figure 6B). Fractionation analysis of nuclear and cytoplasmic proteins of developing maize kernels (15 DAP) showed O11 to accumulate both in the nucleus and the cytoplasm (Figure 6C). A yeast transactivation assay was performed to investigate whether O11 possesses transcriptional activity, and weak transactivation capacity was observed (Figure 6D).
o11 Affects Transcription of Cellular Carbohydrate, Amino Acid Metabolism, and Stress Response Genes
To understand the molecular basis of the phenotypic defects of the o11 mutant, significantly differentially expressed genes (DEGs) were identified from RNA-seq data generated by three independent biological replicates from pooled o11 and wild-type kernels at 15 DAP. According to these RNA-seq data, mutation of O11 significantly alters the expression of 1031 genes (P < 0.05, fold change greater than 1.5), including 337 downregulated and 694 upregulated genes (Supplemental Data Set 2).
Among these DEGs, 575 genes were functionally annotated and classified using the Gene Ontology (GO) database (http://bioinfo.cau.edu.cn/agriGO/) (Figure 7A; Supplemental Data Set 3). Loss of function of O11 significantly affected cellular carbohydrate metabolic process (GO: 0044262, P value: 7.1E-06), and 29 DEGs were identified in this GO classification. The 29 DEGs were functionally clustered into two major categories: carbohydrate catabolic process (18 DEGs) and carbohydrate biosynthetic process (9 DEGs). Approximately 83% DEGs involved in the carbohydrate catabolic process were significantly upregulated. In addition, Brittle-2 (Bt2; GRMZM2G068506) was among DEGs involved in carbohydrate biosynthetic process and was found to be significantly downregulated in o11 kernels (fold change = −1.71). Bt2 encodes the small subunit of maize endosperm ADP-glucose pyrophosphorylase (AGPase), which is considered to be a rate-limiting enzyme in starch biosynthesis (Dickinson and Preiss, 1969; Preiss et al., 1990). Thus, o11 kernels exhibit increased expression of genes functioning in the carbohydrate catabolic process and decreased expression of key gene (e.g., Bt2) in starch biosynthesis.
Regarding amino acid metabolic processes, 30 DEGs (GO: 0006520, P value: 3.7E-06) were functionally clustered into three categories: amino acid biosynthetic process, amino acid catabolic process, and amino acid interconversion. Most DEGs involved in these processes were upregulated, suggesting that o11 has a more active amino acid metabolism compared with the wild type.
Response to stress process was also affected in o11 (GO: 0006950, P value: 0.0011), as 64 DEGs were identified. Over half of the DEGs in this cluster (33 DEGs) were involved in response to cold. Other DEGs were related to multiple types of stress response pathways, such as the response to biotic stress, oxidative stress, or DNA damage stimuli.
Mutation of O11 also affected the processes of nucleosome assembly and translation, which play fundamental roles in developmental processes. The 12 DEGs related to nucleosome assembly (GO: 0006334, P value: 0.0037) included multiple genes encoding histone proteins. In addition, 29 DEGs were related to translation (GO: 0006412, P value: 0.043), including numerous genes encoding ribosomal subunits and elongation factors.
ChIP-Seq Identifies Genes Directly Targeted by O11
To determine which DEGs are directly regulated by O11, chromatin immunoprecipitation sequencing (ChIP-seq) analysis was performed with an O11-specific antibody. The results shown in Supplemental Figure 7 validate the specificity of the O11 antibody. Cross-linked wild-type kernels and o11 mutant kernels at 15 DAP were used for the ChIP-seq assay, and O11 binding sites were identified from consistent peaks generated by two independent biological replicates. A total of 9885 peaks distributed on 6033 genes were identified (Supplemental Data Set 4). We analyzed the distribution of O11 binding sites in the genome. Among all detected peaks, 66.5% were located in genic regions (−1 kb of the transcription start site [TSS] to +1 kb of the transcription termination site [TTS]) and 41% in core promoter regions (−1 kb to +100 bp of the TSS) (Figure 7B). In agreement with its predicted TF function, O11 binding sites were significantly concentrated within the 100 bp immediately upstream of TSSs (Figure 7C).
De novo discovery of the enriched motifs in the binding sites of the genic regions identified the G-box (CACGTG) as the top-scoring motif (52%, E-value = 2.5E-251) (Figure 7D). To test whether the G-box mediates transcriptional regulation of O11, a transient transcription dual-luciferase assay (dual-LUC assay) was performed using the promoters of potential O11 targets (GRMZM2G102605 and GRMZM2G019819) (Supplemental Figures 8B to 8D). These targets were selected based on the presence of the G-box motif under the summit of the O11 binding sites and their downregulation in o11. The same promoters with the G-box replaced by "AAAAAA" were used as a control (Supplemental Figure 8B). O11 significantly activated these promoters, whereas G-box mutation dramatically decreased or eliminated transactivation by O11 (Supplemental Figures 8C and 8D). Our data indicate that O11 can transactivate the promoters of its target genes and that full transactivation depends on the presence of the G-box binding motif.
To explore the direct regulatory function of O11 across the genome, all genes directly bound by O11 in promoter regions (3971 genes) were analyzed using the GO database (http://bioinfo.cau.edu.cn/agriGO/). Among them, 2086 genes were functionally annotated and classified into seven GO terms under four biological processes: metabolic process (cellular carbohydrate metabolic process [GO: 0044262, P value: 6.50E-05]); stress response process (response to stress [GO: 0006950, P value: 6.60E-10] and response to chemical stimulus [GO: 0042221, P value: 1.10E-14]); developmental process (organ development [GO: 0048513, P value: 8.00E-07] and signal transduction [GO:0007165, P value: 4.30E-08]); and gene expression (regulation of transcription [GO:0045449, P value: 1.20-11E] process and posttranslational protein modification [GO:0043687, P value: 2.40E-06]) (Supplemental Figure 9 and Supplemental Data Set 5). To obtain high-confidence targets that are primarily regulated by O11, we compared 3971 genes bound by O11 in the promoter region to the 1031 DEGs from the o11 RNA-seq data. In total, 259 high-confidence potential targets of O11 were identified (Figure 7E; Supplemental Data Set 6).
O11 Directly Regulates Genes Involved in Carbohydrate Metabolism and Stress Responses
To explore the biological functions of O11, the 259 high-confidence potential targets were functionally annotated and classified using the GO database (http://bioinfo.cau.edu.cn/agriGO/), and two GO clusters were generated. One GO cluster was cellular carbohydrate metabolic process (GO: 0044262, P value = 0.041) and was complemented by the MapMan database (Table 1). Starch synthase5 (Ss5) and nine genes involved in glycolysis, the tricarboxylic acid (TCA) cycle, fermentation, and other carbohydrate metabolic process were found to be direct targets of O11, including those encoding pyruvate kinase (ZmPKp-β1), glyceraldehyde-3-phosphate dehydrogenase (Gpc1), and 6-phosphofructokinase and alcohol dehydrogenase 1 (Adh1). Loss of function of O11 significantly downregulated Ss5 and upregulated most carbohydrate metabolic genes (Table 1). Dual-luciferase (LUC) assays using the promoters of down-regulated Ss5 and ZmPKp-β1 in this cluster were performed, and the results showed that O11 specifically activates the Ss5 and ZmPKp-β1 promoters (Figures 8A and 8B). These results demonstrate that O11 directly regulates genes involved in carbohydrate metabolism.
The contents of the intermediate metabolites of glycolysis and the TCA cycle were also analyzed in wild-type and o11 developing endosperm at 15 DAP by gas chromatography-mass spectrometry (GC-MS). The contents of glucose-6-phosphate, citrate, succinate, and fumarate, key intermediate metabolites in glycolysis and the TCA cycle pathways, were significantly reduced in o11 (Figure 9; Supplemental Table 1), suggesting that the loss of O11 function significantly affects glycolysis and TCA metabolism in the maize endosperm.
The other GO cluster identified was response to stress (GO: 0006950, P value = 0.0048), and 21 targets were annotated as stress response genes (Table 1). Target genes involved in this category included proteins of related functions, such as amino acid metabolism, DNA repair, apoptosis regulation, and cytokinin signaling. Four of the six downregulated targets had functional annotations (l-O-methylthreonine resistant1 [ZmOMR1], DNA repair ATPase-related protein [DRAP; GRMZM2G075655], and two low molecular weight cysteine-rich [LCR] 69 proteins [LCR 69a and LCR 69b]). These four targets were further examined in dual-LUC assays, and the results showed that activation of all these promoters was specifically induced by O11 (Figures 8C to 8F). These results demonstrate that O11 directly regulates genes involved in responses to stress.
O11 Regulates Genes Specifically Expressed in the ESR
The ortholog of O11 in Arabidopsis, AtZOU, plays critical roles in the ESR in regulating endosperm breakdown and embryo epidermal development (Yang et al., 2008; Xing et al., 2013; Denay et al., 2014; Fourquin et al., 2016). Yang et al. (2008) showed that zou endosperm fails to separate from the embryo, restricting embryo expansion and resulting in the production of shriveled, collapsed seeds. Analogous to the morphological defects observed in atzou, the o11 ESR displayed similar adhesion to the embryo (Figure 1F). RNA-seq analysis of laser-capture microdissected compartments of the maize kernel (LCM RNA-seq) has been performed, and ESR-specific genes with preferential ESR expression were identified (Zhan et al., 2015). We overlapped the reported ESR-specific genes with the 259 high-confidence potential targets of O11, revealing five ESR-specific genes regulated by O11 (Table 1). One of these genes, ZmYoda, is the ortholog of Arabidopsis YODA (AtYODA), which encodes a mitogen-activated protein kinase kinase kinase (MAPKKK). AtYODA was previously reported to be a key component in the MAPK pathway, regulating embryo differentiation and stomatal development in Arabidopsis (Bergmann et al., 2004; Lukowitz et al., 2004; Wang et al., 2007; Kim et al., 2012). In maize, ZmYoda is significantly downregulated (fold change = −3.1) due to O11 loss of function (Table 1). Another ESR-specific target, ZmSag29, encodes a hexose and sucrose transporter, the transcript of which was increased 5.1-fold in the o11 mutant. ZmSag29 is the ortholog of SENESCENCE-ASSOCIATED GENE29 in Arabidopsis (AtSAG29), and it is reported that overexpression of AtSAG29 accelerates senescence (Seo et al., 2011). Dual-LUC assays using the promoters of down-regulated target genes (ZmYoda and a subtilisin-like protease [Slp]) were performed, and the results demonstrated that O11 can activate the promoters of both ZmYoda and Slp (Figures 8G and 8H). All these data indicate that O11 regulates ESR-specific genes in the maize endosperm.
O11 Regulates Key TFs and Coregulates cyPpdks with O2
Approximately 8% (21 of 259) of the O11 high-confidence potential targets obtained encoded TFs (Table 1), including NKD2, ZmDOF3, PBF, and O2, which are known key TFs that play important roles in both endosperm development and nutrient accumulation. NKD2 regulates a series of biological processes in endosperm development, and ZmDOF3 is a key regulator of starch accumulation and endosperm development (Yi et al., 2015; Gontarek et al., 2016; Qi et al., 2017). O2 and PBF reportedly to play pivotal roles in zein protein and starch accumulation (Vicente-Carbajosa et al., 1997; Wu and Messing, 2012; Li et al., 2015; Zhang et al., 2016). Moreover, Silky1 and ZmWox2 are direct targets of O11 and might function in endosperm development, as SILKY1 was previously shown to be a key regulator of maize floral organ development (Ambrose et al., 2000), and AtWOX2, the ortholog of ZmWOX2 in Arabidopsis, is involved in embryo apical patterning and cotyledon development (Wu et al., 2007; Breuninger et al., 2008; Lie et al., 2012). As downregulated targets, the promoters of the genes encoding ZmDOF3, PBF, SILKY1, and ZmWOX2 were selected for dual-LUC assays, and the results showed all to be specifically induced by O11 (Figures 8I to 8L).
O2 was increased both at the mRNA (1.82-fold) and protein levels in o11 (Figure 10A). To investigate the regulatory relationship between O11 and O2 in the developing maize endosperm, we overlapped all 2564 peaks from the O2 ChIP-seq data (Li et al., 2015) with the total 9885 peaks of O11, with 247 overlapping peaks observed (9.6% overlap ratio for O2; 2.5% overlap ratio for O11) (Figure 10B; Supplemental Data Set 7). A total of 171 of the 247 peaks fell into 169 genes, and 73% of them (123 genes) were bound in the promoter regions (Figure 10C). In total, 101 common targets were bound in promoter regions and expressed in the endosperm according to the RNA-seq data of o11 and o2 (Supplemental Data Set 7). Among them, nine targets were transcriptionally altered in both o11 and o2 (P value < 0.05). Interestingly, seven of the nine targets were downregulated in o2 but upregulated in o11, including both cyPpdk1 and cyPpdk2 (Supplemental Data Set 8), suggesting that O11 and O2 have antagonistic actions in regulating their common targets.
We then performed dual-LUC assays using the promoters of cyPpdk1 and cyPpdk2 (Figures 10D and 10E). O2 strongly activated these promoters, whereas O11 showed no significant activation. When O2 and O11 were both present, transactivation of the cyPpdk1 or cyPpdk2 promoter was significantly lower than when O2 was present alone (Figures 10D and 10E). These results demonstrate that O11 might function as a repressor of O2. According to our ChIP-seq data, the O11 binding sites completely cover the O2 binding sites in both cyPpdk1 and cyPpdk2 (Figure 10F). Yeast two-hybrid (Y2H) assay and luciferase complementation image (LCI) assay results indicated that O11 and O2 do not interact with each other (Supplemental Figure 10). Therefore, O11 possibly prevents O2 from binding to the same binding sites in the promoters of cyPpdk1 and cyPpdk2 in vivo. The repression ability of O11 might occur through binding site competition with other strong activators.
ZmICE1 Coordinates Regulation of Stress Response Genes and ZmYoda with O11
As bHLH TFs tend to form homo- or heterodimers to regulate their targets, a Y2H screening assay was performed to identify O11-interacting proteins. After screening ∼8 million recombinant cDNA clones, we identified 35 putative O11-interacting proteins (Supplemental Table 2). Eighteen proteins with independent positive clones from the Y2H screening were further analyzed by an LCI assay (Figure 11). Six bHLH-type TFs, including Inducer of CBF Expression 1 in maize (ZmICE1 or ZmICEa, GRMZM25173534) (Grimault et al., 2015), as well as five non-TF proteins (two aspartate kinases, two Golgi trafficking-related proteins and one plant-specific protein) passed LCI interacting tests (Figure 11).
AtICE1 in Arabidopsis has been identified as a direct regulator of C-repeat binding factors (CBFs) in the response to cold stress (Chinnusamy et al., 2003). ZmICE1 (ZmICEa) has been reported to have a stronger interaction with O11 (ZmZOU) than the other two homologs (ZmICEb and ZmICEc), and these interactions can transactivate target gene promoters from Arabidopsis (Grimault et al., 2015). Among the direct targets of O11 involved in the response to stress, 13 are annotated as genes related to the response to cold. To assess whether O11 and ZmICE1 can coregulate these 13 cold stress response targets in vivo, ChIP-qPCR was performed using an antibody against ZmICE1 (the specificity and immunoprecipitation effectivity of ZmICE1 antibody are shown in Supplemental Figure 11). Eight targets showed enrichment in ZmICE1 ChIP samples (Figure 12A), suggesting that O11 and ZmICE1 coregulate a portion of cold-stress response genes in vivo. A dual-LUC assay was performed with one of the common cold-stress response targets: ZmOmr1. The ZmOmr1pro-LUC reporter was transiently expressed together with O11 or ZmICE1 or both in N. benthamiana leaves. O11 or ZmICE1 alone was able to activate the ZmOmr1pro-LUC reporter (Figure 12B), the expression level of which was significantly enhanced when O11 was combined with ZmICE1 (Figure 12B). These results suggest that O11 and ZmICE1 can coactivate their common targets.
It is reported that AtICE1 also controls stomatal differentiation (Kanaoka et al., 2008). As AtYODA acts as a molecular switch in stomatal differentiation and O11 interacts with ZmICE1 and directly regulates ZmYoda, we speculated that ZmICE1 might also be involved in regulating ZmYoda. We thus performed ChIP-qPCR with a ZmICE1-specific antibody, and the results showed that ZmICE1 binds to the promoter of ZmYoda (Figure 12C). A dual-LUC assay was performed, and both O11 and ZmICE1 were found to activate the ZmYodapro-LUC reporter alone (Figure 12D). In contrast to the results reported above, the expression level of the ZmYodapro-LUC reporter was not significantly enhanced when O11 and ZmICE1 were coexpressed (Figure 12D), suggesting that they coregulate ZmYoda, but not in a cotransactivational manner.
O11 Is a bHLH TF with Potential New Functions in Maize
The strategies to store nutrients in the seeds differ dramatically between monocotyledonous and dicotyledonous species. In the seeds of monocotyledonous species, nutrients are usually stored in the endosperm, with abundant accumulation until maturation. By contrast, the endosperm in the seeds of dicotyledonous species (such as Arabidopsis) is absorbed by the cotyledons during the course of development, leaving only a single layer of endosperm cells; thus, nutrients are stored in the cotyledons (Olsen, 2004). AtZOU, the ortholog of O11 in Arabidopsis, plays critical roles in endosperm breakdown and embryo epidermal development (Yang et al., 2008; Xing et al., 2013; Denay et al., 2014; Fourquin et al., 2016). Our analysis revealed significant differences between these orthologs in terms of their protein sequence, expressed compartments, and biological function. First, O11 contains an additional 300 amino acids at its N terminus (Supplemental Figure 6). Moreover, except for the bHLH domain, sequence similarity between the orthologs is quite low. Thus, the orthologs are in two clades of the phylogenetic tree (Figure 5A). Second, expression of AtZOU is restricted to the ESR region (Yang et al., 2008), but according to our immunofluorescence analysis, O11 accumulates in both the ESR and the SE (Figure 6A). LCM RNA-seq data (Zhan et al., 2015) (Supplemental Table 3) and qPCR results from another study (Grimault et al., 2015) support our observations.
O11 Is a Central Regulator Linking Maize Endosperm Development, Metabolism, and Stress Response
To explore the biological functions of O11, we investigated global transcriptional changes caused by O11 loss of function and found pleiotropic effects in o11, including cellular metabolic processes, developmental processes and stress responses (Figure 7A). Furthermore, to examine direct targets of transcriptional regulation, genome-wide DNA binding sites of O11 were determined by ChIP-seq analysis in developing maize kernels (Figures 7B to 7E). We identified the G-box as the dominant binding motif for O11 (Figure 7D), which is consistent with previous studies of other bHLH TFs (Oh et al., 2009, 2012; Zhang et al., 2013). We performed electrophoretic mobility shift assays to evaluate the binding ability of O11 to the G-box but failed to observe a band shift (Supplemental Figure 8A). We speculate that this result might be because prokaryotic expression of O11 results in the absence of modifications that are essential for DNA binding, e.g., phosphorylation (Klimczak et al., 1992). O11 exhibits dual activator/repressor activity profiles (Figure 7E). O11 activates its targets via transactivation (Figure 8) and might also coactivate common targets with interacting proteins (e.g., ZmICE1) (Figure 12B). The transcriptional repression ability of O11 might be due to its binding to the same regions of other strong activators (e.g., O2) (Figures 10D to 10F), which is defined as a passive repressor (Krogan and Long, 2009).
Through comprehensive transcriptome and DNA binding site analysis, we found that O11 functions as a central regulator in the gene networks governing maize endosperm development, nutrient metabolism, and stress response (Figure 13). O11 not only regulates genes for key TFs in endosperm development (Nkd2 and ZmDof3) but also controls key regulators of endosperm nutrient metabolism (O2, Pbf, and cyPpdk1 and -2). Moreover, O11 is a direct activator of the upstream MAPKK kinase gene ZmYoda, suggesting that its regulatory function in endosperm development occurs through MAPK signaling. We also investigated the regulatory relationship between O11 and an interacting protein (ZmICE1), and the results showed that they might coregulate endosperm development and stress responses.
O11 Is a Key Regulator in Endosperm Nutrient Metabolism
o11 mutant kernels display a reduced and less vitreous (opaque) endosperm (Figures 1A to 1C). Consistently, the starch granules and PBs in the o11 endosperm were smaller, and the total starch and protein contents per kernel were significantly decreased (Figures 2D and 2E). As starch accounts for ∼75% of the dry weight of maize kernels, the reduced starch accumulation in o11 endosperm should be responsible for its reduced endosperm. Numerous studies on opaque mutants have reported positive correlations between morphological alterations of PBs (o1, o2, o6, o7, and o10) or starch granules (o5) and opaque endosperm (Schmidt et al., 1987; Miclaus et al., 2011; Myers et al., 2011; Wang et al., 2011, 2012, 2014; Yao et al., 2016). Starch granules in normal endosperm cells appear to be tightly interwoven with a proteinaceous matrix largely composed of PBs (Wu and Messing, 2010), and alterations in PB or starch granule morphology would change a compact transparent endosperm into a loose opaque endosperm. Thus, we believe that the morphological changes observed for PBs and starch granules in o11 endosperm cells are responsible for the opaque endosperm of o11.
Loss of function of O11 significantly affects transcription of genes related to cellular carbohydrate and amino acid metabolism, indicating the essential role of O11 in endosperm nutrient metabolism (Figure 7A). We found that O11 directly activates a starch synthase gene and represses those involved in glycolysis, the TCA cycle, and other carbohydrate metabolic process (Table 1); such changes would accelerate starch decomposition, revealing the positive roles of O11 in guaranteeing starch accumulation. O2 and PBF are both well-known TFs regulating expression of zein protein genes (Schmidt et al., 1987; Vicente-Carbajosa et al., 1997), and recent studies indicate that they are also involved in the endosperm's carbon and nitrogen metabolism (Li et al., 2015; Zhang et al., 2016). O11 directly regulates both O2 and PBF (Table 1), suggesting upstream regulatory roles for O11 in endosperm nutrient metabolism. Moreover, the o11 endosperm showed a disrupted C/N ratio (Figure 2G). Previous studies have reported that cyPPDKs are key molecular switches in the endosperm starch/protein balance through inorganic PPi-dependent restriction of starch synthesis as well as through provision of substrates for amino acid synthesis (Sheen, 1991; Prioul et al., 2008; Manicacci et al., 2009). O11 directly binds to the promoters of cyPpdk1 and -2 (Figure 10F), and their expression is significantly altered in O11 loss-of-function mutants (Supplemental Data Set 8), suggesting that O11 is a key modulator in the starch/protein balance. Therefore, O11 is involved in multilayer transcriptional regulation of endosperm carbohydrate and amino acid metabolism. Indeed, mutation of O11 disrupts carbohydrate and amino acid metabolism, thereby affecting the accumulation and morphology of starch granules and PBs and, ultimately, resulting in the reduced and opaque endosperm of the o11 mutant.
O11 Is a Key Regulator in Endosperm Development
Loss of function of O11 results in a reduced endosperm with developmental defects (Figure 1), indicating the critical roles of O11 in endosperm development. Our data show that O11 occupies the central position of a regulatory network in endosperm development. In addition to NKD2, ZmDOF3, and SILKY1, the three TFs encoded by the target genes of O11 (ZmWOX2, ZmSVP, and ZmAGL19) might also be involved in endosperm development (Table 1), as their orthologs in Arabidopsis are involved in embryo development, floral meristem differentiation, and floral transition, respectively (Hartmann et al., 2000; Schönrock et al., 2006; Wu et al., 2007; Breuninger et al., 2008; Lie et al., 2012; Gregis et al., 2013). Moreover, two O11-interacting bHLH TFs (ZmBIM and ZmLHW) may also regulate endosperm development (Supplemental Table 2), as their orthologs are reported to function in the brassinosteroid signaling pathway and in root differentiation, respectively (Ohashi-Ito and Bergmann, 2007; Cifuentes-Esquivel et al., 2013).
Previous studies have demonstrated that loss of AtYODA function results in abnormal embryo differentiation and excess stomata in the epidermis, whereas overexpression of AtYODA suppresses embryonic development and eliminates stomata (Bergmann et al., 2004; Lukowitz et al., 2004; Wang et al., 2007; Kim et al., 2012). Thus, the YODA/MAPK cascade plays a major role in regulating both the polarity of asymmetric cell divisions of stomata and zygote development. Our data showed that O11 is a direct activator of ZmYoda (Figure 8G) and that loss of O11 function significantly downregulates ZmYoda transcription (Table 1), suggesting that the regulatory function of O11 in endosperm development occurs through the MAPK pathway. It has been reported that the number of fully differentiated stomata is reduced when O11 is ectopically expressed in maize leaves (Grimault et al., 2015). The decrease in the number of stomata in O11-overexpressing leaves might also be due to excess transactivation of ZmYoda by O11. ZmYoda was found to be preferentially expressed in the ESR (Zhan et al., 2015) (Supplemental Table 3), suggesting that ZmYODA might also be involved in ESR differentiation by regulating asymmetric cell divisions.
The Role of O11 in Stress Responses
In addition to the central roles of O11 in regulating endosperm development, we unexpectedly found that O11 also participates in stress response regulation. Loss of function of O11 caused excessive transcriptional changes in stress response genes (Figure 7A). Moreover, target analysis showed that O11 directly regulates many stress response genes (Table 1), and many TF gene targets of O11 might be involved in the stress response (Table 1), such as ZmJaz1 and ZmWrky40, which, according to the annotations of their orthologs from Arabidopsis, are considered to modulate stress responses (Hu et al., 2013; Xu et al., 2006).
A transcriptome study of the maize developing endosperm reported that stress response TFs (e.g., WRKY family TFs) are differentially expressed under normal conditions during endosperm development (Qu et al., 2016). AtICE1 is a key regulator in the response to cold stress and plays critical roles in stomatal development (Chinnusamy et al., 2003; Kanaoka et al., 2008). Both of these previous studies imply a potential link between the transcriptional regulation of environmental adaptation and development in plants. O11 physically interacts with ZmICE1 (Figure 11; Supplemental Table 2) and coregulates stress response genes and ZmYoda with it (Figure 12), suggesting that O11 and ZmICE1 might coregulate stress responses and endosperm development. We also observed tight correlations between endosperm development and stress responses while investigating o11. Plants accumulate abundant free proline under abiotic stress, such as salinity, drought, or extreme temperature (Verbruggen and Hermans, 2008; Szabados and Savouré, 2010). The endosperm of the o11 mutant contains more than four times the free proline content of the wild type (Supplemental Figure 4B), suggesting that the mutant endosperm is in a state similar to that of abiotic stress. The significant developmental compromise of o11, together with the drastic stress response, suggests that O11 might function as a "balancer" between endosperm development and the stress response.
Plant Material
The o11 mutant stock (o11-ref) was obtained from the Maize Genetics Cooperation Stock Center (accession number ox-7455). The o11 mutants were crossed into a W22 genetic background and then self-pollinated to produce F2 populations or backcrossed to the o11 mutants to produce BC1F1 populations. Kernels from F2 ears were used for cytological and biochemical analyses; the BC1F1 population was used for fine-mapping of o11. Root, stem, third leaf, tassel, silk, husk, and ear tissues were collected from five W22 plants at the V12 stage. Immature kernels of W22 plants were harvested at 3, 5, 9, 12, 15, 18, 21, 24, and 27 DAP. All plants were cultivated in a field at the Shanghai University campus in Shanghai, China.
Measurement of Starch, Protein, Lipid, C/N, and Amino Acid Contents
Twenty mature kernels of the wild type or o11 mutants from the same ears were collected and soaked in water, after which the endosperm was separated from the embryo and pericarp. The wild-type and o11 mutant endosperm tissues were pooled separately, ground in liquid N2, and dried to a constant weight for subsequent analysis.
Total starch was measured using an amyloglucosidase/α-amylase starch assay kit (Megazyme) according to the manufacturer's protocol and adapted as follows. A 0.2-mL aliquot of aqueous ethanol (80% [v/v]) was added to a 100-mg sample to aid dispersion, and 3 mL thermostable a-amylase was immediately added. The tube was incubated in a boiling water bath for 6 min and placed in a bath at 50°C; 0.1 mL amyloglucosidase was then added. The tube was stirred with a vortex mixer and incubated at 50°C for 30 min. Duplicate aliquots (0.1 mL) of the diluted solution were transferred to a new tube; 3.0 mL of glucose oxidase/peroxidase (GOPOD) reagent was added, and the tubes were incubated at 50°C for 20 min. The absorbance of each sample and the d-Glc control at 510 nm was measured against the reagent blank.
Total protein, zein and nonzein protein, lipid, total amino acid, and free amino acid contents were measured according to a previously described protocol (Wang et al., 2011). For carbon and nitrogen content measurements, a Vario EL III Element Analyzer was used with the recommended settings.
Each assay above was independently performed three times on the kernels collected from three different ears
Scanning Electron Microscopy and Transmission Electron Microscopy
For scanning electron microscopy, mature wild-type and o11 kernels harvested from the same self-pollinated ear were analyzed according to a previously described method (Wang et al., 2011). The samples were then observed by scanning electron microscopy (S3400N; Hitachi). For transmission electron microscopy, o11 and wild-type developing kernels (18 DAP) from the same self-pollinated ear were treated and observed as described previously (Wang et al., 2011). The samples were observed with a Hitachi H7600 transmission electron microscope.
Light Microscopy and Immunofluorescence Assay
For light microscopy analysis, immature wild-type and o11 mutant kernels at 12, 15, and 18 DAP were collected from the same ear and cut along the longitudinal axis for paraffin section preparation and along the horizontal axis for resin section preparation. The procedures were performed as described (Chen et al., 2017).
For the immunofluorescence assay, o11 mutant kernels with no accumulation of O11 protein were used as the negative control. Sections (8 μm thick) of paraffin-embedded samples were rehydrated and blocked with 5% BSA in PBS at pH 7.4 (blocking buffer) and incubated with an O11-specific antibody (dilution of 1:100 in blocking buffer). The samples were then incubated with an anti-rabbit-conjugated fluorescein isothiocyanate antibody (Abcam; ab6717, 1:200) and observed by confocal laser scanning microscopy (LSM710; Zeiss).
Retrotransposon Amplification, Complementation Test, and Allelism Test
The retrotransposon was amplified by DNA polymerase (Toyobo; KOD-401) using a nested PCR strategy. Briefly, PCR was performed with o11 homozygous mutant genomic DNA using primers retro-1s and retro-1a. Then the first-round PCR product was diluted 100 times and was used as the second-round PCR template (primers were retro-2s and retro-2a) (Supplemental Data Set 9).
For functional complementation tests, genomic DNA (8464 bp) containing the entire coding region, a 2311-bp upstream sequence, and a 1019-bp downstream sequence was amplified from the B73 inbred genome using the primers listed in Supplemental Data Set 9. The fragment was cloned into pTF102 (Frame et al., 2002), and the resulting construct was introduced into Agrobacterium tumefaciens strain EHA105 (Frame et al., 2002). Agrobacterium-mediated maize (Zea mays) transformation using F2 immature zygotic embryos of the Hi-II hybrid (pApB) was performed according to Frame et al. (2002). Transgenic lines (T0) were hybridized to the homozygous mutant (o11/o11) and then self-pollinated to harvest O11-functionally complemented ears. F/R6, a marker tightly linked to the o11 locus, was used to identify homozygous o11 kernels as PCR products amplified from o11 homozygous genomic DNA using F/R6 primers were larger than those from pApB genomic DNA.
For the allelism test, a CRISPR-Cas9 vector of GRMZM2G147685 was constructed as described using a simplex editing strategy (Qi et al., 2016a). The 20-bp target sequence for editing is located in the first exon of O11 (the 20 bp target sequence is GGGCAACGCCGCCGCGGTGG). Agrobacterium-mediated maize transformation was performed as described above. Five independent Cas9-edited knockout alleles (named o11-cas9-1 to o11-cas9-5) were recovered, and all were separated from the Cas9 transgene.
Phylogenetic Analysis
Related sequences were identified in the NCBI nr (nonredundant protein sequences) database by performing a BLASTp search with O11 protein sequences. Amino acid sequences were aligned with MUSCLE in the MEGA5.2 software package using the default settings for protein multiple alignment. Evolutionary distances were computed using Poisson correction analysis. The bootstrap method with 1000 replicates for phylogeny testing was used.
Polyclonal Antibody Generation and Immunoblot Analysis
For production of an anti-O11 antibody, the full-length O11 cDNA was cloned into the EcoRI and XhoI sites of the pET-32a vector (Novagen) (primers used were 147685-orf-ecori-s and 147685-orf-xhoi-a) and transformed into BL21 cells. For anti-ZmICE1 antibody production, the full-length ZmIce1 cDNA was cloned into the BamHI and XhoI sites of the pET-32a vector (Novagen) (primers used were 173534-orf-bamhi-s and 173534-orf-xhoi-a), which was then transformed into BL21 cells. The recombinant O11- or ICE1-6xHis fusion protein was purified using the ÄKTA purification system with a 1-mL HisTrap FF crude column (GE Healthcare). Both antibodies were prepared in rabbits by Abclonal of China. The primers are listed in Supplemental Data Set 9.
Total protein from wild-type and mutant endosperms was extracted according to the method of Bernard et al. (1994). The protein extracts were quantified by the Bradford assay (Bradford, 1976). Immunoblot analyses were performed as described previously (Wang et al., 2014) using antibodies against the following proteins: O11 (1:1000 dilution), tubulin (Sigma-Aldrich; T6074-200UL; 1:2500 dilution), BiP (Santa Cruz Biotechnology; sc-33757; 1:2000 dilution), H3 (Sigma-Aldrich; H0164-200UL; 1:1000 dilution), and O2 (1:1000 dilution) (Li et al., 2015).
Subcellular Localization of O11 in Nicotiana benthamiana and Fractionation of Maize Seed Cytoplasmic and Nuclear Proteins
The open reading frame (ORF) of O11 was cloned into pENTR/D-TOPO (primers used were O11-topo-s and O11-topo-a listed in Supplemental Data Set 9) using the Gateway TOPO cloning kit (Invitrogen). The right entry clone was introduced into the pB7WGY2 plant expression vector through the LR reaction of the Gateway system (Invitrogen). The expression vectors were transformed into Agrobacterium strain GV3101 as previously described (Wydro et al., 2006). The eYFP signal was observed and imaged using a confocal microscope (LSM710; Zeiss). Fractionation of cytoplasmic and nuclear proteins of 15-DAP maize immature kernels was performed using a previously described method (Qi et al., 2016b).
RNA-Seq and qPCR
Total RNA was extracted from pooled wild-type or o11 mutant kernels obtained from the same F2 ear at 15 DAP (20 kernels each sample) using Trizol reagent, followed by cleanup and DNase I treatment with the Qiagen RNeasy mini kit in accordance with the protocol provided with the kit. Three independent biological replicates from three different ears were performed. cDNA libraries were constructed following Illumina standard protocols and sequenced with an Illumina HiSeq 2500 by Shanghai Biotechnology. The sequence reads were trimmed using fastx (version: 0.0.13) and mapped to B73 RefGen_v3.24 using TopHat (version: 2.0.9). Cufflinks (version: 2.1.1) was employed to reconstruct the transcripts and to estimate gene expression levels (Trapnell et al., 2010). Significant DEGs were identified as those with a P value of differential expression above the threshold (P < 0.05, fold change greater than 1.5). GO enrichment was performed with the accession numbers of significant DEGs via agriGO Singular Enrichment Analysis tool with default parameters (http://bioinfo.cau.edu.cn/agriGO/).
RT-qPCR was performed with SYBR Select Master Mix (Thermo) using a Mastercycler ep realplex 2 (Eppendorf). Levels were calculated using the ΔCt (threshold cycle) method and the maize Actin gene was used as the reference. All primers used in the study are listed in Supplemental Data Set 9.
ChIP-Seq and ChIP-qPCR
For ChIP-Seq, ChIP using an antibody against O11 with pooled W22 or o11 mutant kernels at 15 DAP was performed as previously described (Kaufmann et al., 2010; Li et al., 2015). Two independent biological replicates prepared at different times were performed. Briefly, dissected 15 DAP kernels were immediately cross-linked in buffer containing 1% formaldehyde on ice under vacuum for 15 min, after which the vacuum was released and reapplied for another 14 min. Fixation was stopped by adding 1.25 M glycine. The cross-linked kernels were dried and ground into powder in liquid N2, followed by nuclear isolation and sonication. Protein A-agarose beads (Millipore) and an antibody against O11 were used to precipitate the DNA, which was digested by proteinase K, recovered using a QIAquick PCR purification kit (Qiagen) and analyzed by qPCR (Supplemental Data Set 9). ChIP-DNA libraries were constructed and sequenced by Shanghai Hanyu Bio-Tech. Briefly, DNA samples were end-repaired, followed by A-base addition and ligation with adapters using TruSeq PE Cluster Kit (Illumina). After PCR enrichment, the DNA library was quantified with Qubit (Thermo Fisher), followed by cBot cluster generation (Illumina) and library sequencing with an Illumina HiSeq 2500.
ChIP was performed using the same protocol as for ChIP-seq. For ChIP-qPCR of ZmICE1, a ZmICE1-specific antibody was used to precipitate the chromatin; IgG was used as an antibody control. In brief, the level of each gene in three biological replicates was normalized to that of Actin measured in the sample with SYBR Select Master Mix (Thermo) using a Mastercycler ep realplex 2 (Eppendorf). Levels were calculated using the ΔCt (threshold cycle) method. The primers used for ChIP-qPCR are listed in Supplemental Data Set 9.
ChIP-Seq Data Analysis
Raw fastq data were trimmed using the SolexaQA++ v2.5 program with Q20 (Phred score >20) and L40 (length >40). The cleaned reads were then aligned to the maize B73 reference genome (AGPv3; http://plants.ensembl.org/) using Bowtie2 (v2.3.2) with default parameters (Langmead et al., 2009). Unique aligned reads were extracted by the SAMtools v1.2 program with a MAQ20 (map quality >20) filtration. Peak calling was performed separately for each biological replicate using MACS2 (v2.1.0) (bandwidth = 300; model fold = [10, 30]; q-value cutoff = 0.01) (Zhang et al., 2008). Significant peaks with high confidence in both biological replicates were considered to be consistent peaks. To search for conserved binding motifs in consistent O11 binding regions, the 500-bp sequence surrounding the peak summit of each consistent peak was extracted and submitted to the online version of MEME-ChIP (Machanick and Bailey, 2011) under default settings. A custom background model derived from JASPAR CORE (2016) plants was provided, and "any number of repetitions" of a motif was allowed.
Metabolite analysis by GC-MS was performed according to a described method, with the following minor modifications (Fait et al., 2006). Briefly, 250 mg maize endosperm was homogenized in liquid N2 and extracted with methanol:chloroform:water extraction solution (900 μL, 1:2.5:1 v/v). A standard (0.2 mg mL−1 ribitol in water) was added as for quantification. The mixture was extracted for 15 min at 4°C with shaking. After centrifugation at 2200g for 2 min, 400 μL water was added to the supernatant. Following vortexing and centrifugation, the methanol-water phase was collected, and 300 μL was reduced to dryness under vacuum. The residue was redissolved and derivatized for 90 min at 37°C (in 60 μL 20 mg mL−1 methoxyamine hydrochloride in pyridine) followed by a 30-min treatment with 90 μL N-methyl-N-(trimethylsilyl)trifluoroacetamide at 37°C. GC-MS analysis was performed using an Agilent 6890 gas chromatograph coupled to a 5975C inert MS detector (Agilent Technologies) equipped with a capillary column (HP-5MS) with an internal diameter of 250 μm, a length of 30 m, and a film thickness of 0.25 μm. Helium was used as the carrier gas at a flow rate of 1 mL/min. The injection volume was 5 μL. Splitless injection mode was used for all samples. The optimized GC-MS front inlet temperature was 220°C. Oven temperature was set at an initial temperature of 60°C, held for 2 min, increased to 180°C at 5°C/min, and then increased to 260°C at 10°C/min, where it was held for 5 min. The instrument was operated using an electron ionization source at 70 eV, and the MS source and quad temperatures were 230°C and 150°C, respectively. The scanning range was 40 to 700 (mass-to-charge ratio). Products were identified by comparison with standards and the NIST (National Institute of Standards and Technology) library. Peak areas of wild-type and o11 metabolites were normalized by the internal standard ribitol, which was derivatized concomitantly with the tissue samples, and were compared with the mean peak area calculated from the corresponding wild type. Three biological replicates were produced from three independently pooled wild-type or o11 endosperm. For peak area of the carbohydrate metabolites, see Supplemental Table 1.
Y2H Assay and Yeast Transactivation Assay
The full-length ORF of O11 was fused to the GAL4 BD domain in pGBKT7 at the NcoI and EcoRI sites using primers O11-ncoI-orf-s and O11-ecori-orf-a (Supplemental Data Set 9). We used this bait to screen a maize developing kernel cDNA library fused downstream of the GAL4 AD domain in pGADT7 (Wang et al., 2010). The O2 ORF was fused to pGADT7 at the XmaI and ClaI sites. The yeast transactivation assay was performed as previously described (Qiao et al., 2016). The full-length O11 cDNA was cloned into the pGBK-T7 vector and co-transformed into the EGY48 yeast strain with the pG221 vector. The pG221 vector contains a minimal promoter that is bound by the DNA binding motif encoded by the pGBK-T7 vector. The empty pGBK-T7 vector was used as a negative control. pGBK-T7-O2 containing the full-length O2 ORF was used as a positive control.
Transient Transcription Dual-LUC Assay
Dual-LUC assays using N. benthamiana plants were performed as described previously (Hellens et al., 2005; Liu et al., 2008). The luciferase activity of the plant extract was analyzed by a luminometer (Promega 20/20) using commercial LUC reaction reagents according to the manufacturer's instructions (Promega). For dual-LUC assays, three independent experiments at different times (biological triplicates) were performed.
For the effectors used in this study, the full-length ORF of O11 or ZmICE1 was fused into the overexpression vector (pHB) (Yao et al., 2016) using primers O11-hindiii-ORF-S and O11-saci-ORF-A, or 173534-orf-bamhi-s and 173534-ORF-xbai-A, respectively. For the reporters, the promoters of O11-potential targets (about −1 kb to 0 bp of start codon) were cloned into pGreenII0800-LUC (the specific restriction enzyme sites and primers used are indicated in Supplemental Data Set 9).
LCI Assay
The ORFs of O11 and O2 and the 18 potential genes encoding O11-interacting proteins identified from Y2H screening were cloned into both JW771 (NLUC) and JW772 (CLUC) (Zhang et al., 2015) using ClonExpress II One Step Cloning Kit (Vazyme) (the primers are listed in Supplemental Data Set 9). The constructs were transformed into Agrobacterium strain GV3101. The Agrobacterium (strain GV3101) cells were grown to OD600 = 0.8, pelleted, and resuspended in infiltration buffer (10 mM methylester sulfonate, 10 mM MgCl2, and 150 µM acetosyringone, pH 5.7) and infiltrated into 3-week-old N. benthamiana leaves using a needleless syringe in the different combinations indicated in Figure 11. After 48 h under the condition of 16 h of light and 8 h of dark, leaves were injected with 1 mM luciferin (Promega). The resulting luciferase signals were collected using the Tanon-5200 image system. The LCl assay was performed three times independently.
Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: O11, DAA37785.1, GRMZM2G147685; ZmICE1, NP_001131774.2, GRMZM2G173534; ZmYODA, NP_001308276.1, GRMZM2G093316; O2, XP_008651859.1, GRMZM2G015534; PBF, NP_001105400.2, GRMZM2G146283; cyPPDK1, AFW77961.1, GRMZM2G306345; cyPPDK2, XP_008656296.1, GRMZM2G097457; NKD2, NP_001159309.1, GRMZM5G884137; ZmDOF3, NP_001131758.1C, GRMZM2G137502; AtZOU, NP_175399.2, AT1G49770; AtICE1, NP_189309.2, AT3G26744; AtYODA, and NP_176557.1, AT1G63700. Sequences used for phylogenetic analysis are as follows: Sorghum bicolor, KXG26314.1; Setaria italica, KQK97270.1; Oryza sativa japonica group, XP_015635110.1; Brachypodium distachyon, XM_003572553.1; Aegilops tauschii, KD581806.1; Hordeum vulgare subsp Vulgare, dbj|AK373155.1; Arabidopsis thaliana (AtZOU), OAP16519.1; Brassica napus, XP_013752539.1. Populus euphratica, XP_011000121.1; Vitis vinifera, XP_002282369.1; Solanum tuberosum, XP_015163703.1; Nicotiana tabacum, XP_016454613.1; Gossypium raimondii, XP_012466617.1; Manihot esculenta, OAY50956.1; Ricinus communis, EEF52378.1; and Physcomitrella patens, XP_001778019.1. RNA-seq and ChIP-seq data are available from National Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under the series entries GSE97422 and GSE102051.
Supplemental Data
Supplemental Figure 1. Phenotype and dry weight analysis from kernels of o11xW22 F2 ears.
Supplemental Figure 2. Paraffin sections of 18 DAP wild-type and o11 kernels for full-kernel images.
Supplemental Figure 3. Comparison of starch, protein (total protein, zein, and nonzein), and lipid accumulation in the mature wild-type and o11 endosperm according to weight.
Supplemental Figure 4. Analysis of total amino acid and free amino acid contents of the wild type and o11 mature endosperm.
Supplemental Figure 5. GRMZM2G147685 knockout alleles and allelism test with o11.
Supplemental Figure 6. Diagram showing the O11 protein sequence and its homologs in monocotyledonous species (Sorghum bicolor, Setaria italica, and Oryza sativa japonica group) and dicotyledonous species (Arabidopsis thaliana and Brassica napus).
Supplemental Figure 7. Determination of anti-O11 antibody specificity.
Supplemental Figure 8. EMSA assay and transient transcription dual-luciferase (dual-LUC) assays using the promoters of O11 target genes with the wild type (Gwt) and mutant version (Gmu) of the G-box (CACGTG).
Supplemental Figure 9. GO classification of genes with promoter regions that are directly bound by O11, as identified by ChIP-seq.
Supplemental Figure 10. O11 does not interact with O2 in a yeast two-hybrid assay or an LCI assay.
Supplemental Figure 11. Determination of the ZmICE1 antibody specificity.
Supplemental Figure 12. Full scan images of immunoblots.
Supplemental Table 1. Peak area of the carbohydrate metabolites of wild type and o11 endosperm.
Supplemental Table 2. List of O11-interacting proteins, as identified by a Y2H assay.
Supplemental Table 3. Normalized expression levels in FPKM for O11, ZmIce1, and ZmYoda in different compartments of the maize kernel from LCM RNA-seq data (Zhan et al., 2015).
Supplemental Data Set 1. Text file of the alignment used for the phylogenetic analysis shown in Figure 5A.
Supplemental Data Set 2. Significantly differentially expressed genes identified from RNA-seq data for o11 at 15 DAP.
Supplemental Data Set 3. Gene Ontology classifications of o11 DEGs with functional annotations.
Supplemental Data Set 4. O11 binding sites identified by ChIP-Seq.
Supplemental Data Set 5. Gene Ontology classifications of O11 ChIP binding sites (promoter region) with functional annotations.
Supplemental Data Set 6. The 259 high-confidence potential targets of O11.
Supplemental Data Set 7. Overlapping peaks between O11 and O2 across the genome.
Supplemental Data Set 8. Genes bound by both O11 and O2 in 15-DAP developing endosperm.
Supplemental Data Set 9. List of primers.
Supplemental File 1. Student's t test tables.
We thank Lin Xu (Shanghai Institute of Plant Physiology and Ecology, SIBS) for technical support with ChIP experiments. We thank Yanan Feng and Huiling Ling for technical support with Y2H and maize transformation experiments. This work was supported by the National Natural Sciences Foundation of China (91635303), the National Key Research and Development Program of China (2016YFD0101003), and the National Natural Science Foundation of China (31425019, 91335208, and 31401386).
R.S., F.F., and W.Q. designed the research. F.F., S.Y., L.X., W.Y., and Y.C. performed the experiments. F.F., R.S., W.Q., Y.L., and H.Z. analyzed the data. F.F., R.S., and W.Q. wrote the article.
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.plantcell.org) is: Rentao Song (rentaosong{at}cau.edu.cn).
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- Received August 2, 2017.
- Revised January 22, 2018.
- Accepted February 6, 2018.
- Published February 7, 2018.

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Create fantastic party decor for any birthday, sports or fundraising event with this Volleyball Mylar Balloon. Attach Volleyball Balloons to chair backs, create balloon centerpieces or use curling ribbon and a balloon weight to place them throughout the party.
Please allow ample time for delivery. The delivery date for this product is noted above for US shipping only. Please refer to checkout for delivery dates outside of the Contiguous 48 States.
Shipping charges are based on the value of the merchandise and not the number of shipments. For additional shipping information, please contact our Customer Service Department at 800-348-5084.


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Essential Enzymes with Probiotics!
Enzyme deficiencies may result from a combination of age' diet and lifestyle. These deficiencies can lead to a variety of digestive discomforts' including occasional gas' bloating' irregularity and indigestion.
Fortifying the digestive system with enzymes and probiotics may promote optimal digestion while maintaining the digestive system's natural balance to support overall health.
Digest™ Basic +Probiotics contains a gentle enzyme formula that breaks down carbohydrates' fats' fiber and protein. Probiotics provide additional digestive support by boosting enzyme production and promoting regularity.
Product Highlights:
- Gentle support for overall digestive health.
- 750 million active cultures per daily dose.
These statements have not been evaluated by the Food and Drug Administration (FDA). These products are not meant to diagnose' treat or cure any disease or medical condition. Please consult your doctor before starting any exercise or nutritional supplement program or before using these or any product during pregnancy or if you have a serious medical condition.
Serving Size: 1 Capsule
Servings Per Container: 30
Amount Per Serving / % DV
Protease Thera-Blend™ 20'000 HUT / **
Amylase Thera-Blend™ 8'440 DU / **
Pectinase 25 Endo-PGU / **
Maltase 100 DP° / **
Cellulase Thera-Blend™ 500 CU / **
Glucoamylase 10 AGU / **
Alpha Galactosidase 50 GaIU / **
Lactase 300 ALU / **
Lipase Thera-Blend™ 450 FCCFIP / **
Invertase 270 SU / **
Xylanase 100 XU / **
Probiotic Blend: 250 million CFU / **
L. acidophilus DDS-1' L. rhamnosus' L. casei' L. gasseri' L. plantarum' L. bulgaricus' L. salivarius' L. paracasei.
**Daily Value not established.
Other Ingredients: 100% vegetarian capsule (cellulose' water).
Contains no egg' dairy' preservatives' salt' sucrose' soy' wheat' yeast' nuts' corn' gluten' casein' potato' rice' artificial colors or flavors.
Non-GMO / Vegetarian / No fillers added.
Made in USA.
1 capsule with each meal. More may be taken as needed. For higher enzyme potency' Enzymedica's Digest™ +Probiotics or Digest Gold™ +Probiotics may be helpful.
Keep closed in dry place; avoid excessive heat. Please keep out of reach of children. Do not use if safety seal is broken or missing.
Allergy / Dietary Needs
- Corn Free
- Dairy Free
- Egg Free
- Gluten Free
- GMO Free
- Made in the USA
- Nut Free
- Salt Free
- Soy Free
- Vegetarian
- Wheat Free
- Yeast Free


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Check out these Long Island fairs and festivals for the month of August! Get in those activities before it's back to school!
Sayville Summerfest 8/6-8/7
Holbrook Carnival/Craft Festival 8/11-8/14
Long Island Sound & Art Festival – Northport Harbor 8/7
The Long Island Bluegrass Festival – Town of Babylon 8/20
The 2016 Polish Town Fair – 8/20-8/21
The 42nd Annual Polish Town Fair will be held August 20-21 in Polish Town, USA.
Free Street Fair & Carnival from 10am to 6pm both days.
Booths featuring a variety of food, crafts, imports, arts, jewelry and specialties.
10am: Polish Mass at St. Isidore's RC Church, followed by a reenactment of an old fashioned Polish Wedding at noon on the church steps.
11:30am: Flag raising ceremony by the show mobile with a Polish choir, plus demonstration of Polish customs.
Miss Polish Town USA 2016 will reign over festivities. Come taste Polish Town's famous kielbasa sandwiches, pierogi and grandma's polish babka bread. Don't forget to pick up a souvenir cook book, t-shirt and annual 2016 Polish Town mug! Music both days at the show mobile will be provided by DJ Hank.
POLISH DANCE FESTIVAL AT POLONAISE PARK! 6 hours of polka fun starting at 5pm. Music provided by The Boys along with Mike Costa and the Beat. Admission $13 for adults, children under 12 $8.
Sunday: Join us under our Pavilion for family entertainment beginning at noon.
Tomato Fest – Riverhead 8/27-8/28
Saturday and Sunday August 27 and 28, 10am to 6 pm rain or shine
Come judge the taste of 20 varieties of heirloom and traditional tomatoes. Learn to can your own, make salsa, and enjoy tomato cuisine!
Enjoy a full schedule of events including U-Pick Cherry Tomatoes and hayrides, and workshops on "How to Grow Tomatoes" and "Tomato Canning"
Great for adults and kids alike. GROW. EAT. ENJOY!
The 53rd Annual Art Show & Craft Fair – Shelter Island 8/27
sponsored by the Shelter Island Chamber of Commerce will be held on Saturday, August 27th, 2016, 10 a.m. – 5 p.m. at the Shelter Island School 33 North Ferry Road, Shelter Island.
It's our 53rd year. Join quality artists and craftspeople in our well-attended show that is open to artisans only to display, demonstrate and sell their outstanding, high quality, original works in any medium or style.
There is also a special youth category and a Shelter Island section for SI Chamber members only.
This event will be held on the grounds of the Shelter Island School
on Rt. 114, with free admission, free parking and draws thousands of attendees.
There is also a vendor-donated raffle raising funds for high school scholarships.
So apply early – limited spaces.
August 27, 2016, 10 a.m. – 5 p.m.
Great Exposure – Great Sales – Great Community Support
Vendor Donated Raffle · Artist Showcased Publicity Poster · Special Youth Category
Links Festival – Calverton 8/28
Five bands, live painting, gourmet food trucks and children's activities! Calverton Links, Calverton. $25 in advance, $35 at gate, $10 Ages 6-15, Free 5 & under. 12-8 p.m. Aug. 28.


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In This Section
Mitochondria Affect How We Respond to Stressful Environments
Feeling frazzled and run down seems as synonymous as mistletoe and holly with the holidays. New research suggests that the tiny structures inside our cells that generate energy, called mitochondria, may play a role in our mind-body interactions and how we respond to stressful environments, such as jam-packed shopping malls.
Douglas C. Wallace, PhD, a pioneering scientist in mitochondrial medicine and director of the Center for Mitochondrial and Epigenomic Medicine at The Children's Hospital of Philadelphia, has led research in animals showing how alterations in mitochondrial function lead to distinct physiological changes in hormonal, metabolic, and behavioral systems in response to mild stress. While researchers have long recognized individual differences in response to environmental cues such as stress, identifying the genetic and physiological basis for these individual differences has eluded scientists.
The researchers subjected the mice to a standardized psychological stress: placing them in restraint for a brief period. They then measured the effects of this stressor on the animals' neuroendocrine, inflammatory, metabolic, and gene transcription systems. In humans, all of these systems are involved in behavioral responses to stress. Frequently activating stress responses can inflict long-term damage in mammals and humans. The cumulative wear and tear on the body can result in both psychological disorders and human diseases such as diabetes and age-related cognitive decline.
In the mice, relatively mild mutations in mitochondrial genes, located in either mitochondrial DNA (mtDNA) or nuclear DNA, produced unique whole-body stress-response signatures, indicated by physiological and gene expression patterns. mtDNA code for essential energy genes and exchange biological signals with the more familiar DNA housed in the cell nucleus. Those interactions affect physiological networks essential for health.
"This has profound implications for the hereditary basis of neuropsychiatric diseases and for the role of stress in human health," Dr. Wallace said of the study that he published with colleagues in Proceedings of the National Academy of Sciences.
Although he emphasized that much more research remains to be done on the role of mitochondria on human behavior, Dr. Wallace postulated that the study indicates that an important reason for our limited progress in understanding the genetic and biologic basis of psychology is our lack of appreciation for the importance of systematic alterations in energetic metabolism. Dr. Wallace pointed out that while the brain constitutes only 2 percent of human body weight, it consumes 20 percent of the body's energy; therefore, mild variations in mitochondrial bioenergetics could have significant effects on the brain.
"Scientists have long known that stressful experiences, on their own, do not cause disease; it's our responses to stress that have the potential to culminate in disease," Dr. Wallace and colleagues stated. "In this emerging paradigm, mitochondria are at the interface of genetic and environmental factors contributing to disease trajectories."
Identifying the altered mitochondrial states associated with neuropsychiatric diseases may help suggest new therapies. These may permit physicians to more effectively ameliorate the effects of environmental stressors on human health and help people become more resilient in environmental changes.
In addition to his CHOP position, Dr. Wallace is on the faculty of the Perelman School of Medicine at the University of Pennsylvania. His co-authors are from CHOP, Penn, Thomas Jefferson University, and Rockefeller University.
For more information, see the related press release.


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VBAC Facts
Avoid a repeat C-section.
(page 1 of 2)
"Go for it!" is not the advice pregnant women contemplating a VBAC — or a vaginal birth after a previous C-section — are accustomed to hearing. But that's exactly what the medical director of Christiana Care Women's Health Ambulatory Services in Wilmington tells many of her expectant moms.
"In a well-selected patient, VBAC is very beneficial," says Elizabeth Zadzielski, MD. Indeed, the National Institutes of Health reports that 60 to 80 percent of women who attempt a VBAC are able to successfully deliver vaginally.
"The majority of women would likely be candidates" for a VBAC, concurs Sindhu K. Srinivas, MD, director of obstetrical services at the Hospital of the University of Pennsylvania. And according to the American Congress of Obstetricians and Gynecologists, a successful VBAC has fewer complications than an elective repeat Cesarean. Given this fact, why don't more women attempt VBACs?
"There are a lot of misconceptions out there," says Ginnie Calabrese, co-founder of Loving Birth Services in Williamstown, NJ. Jennifer Gleeson Blue, co-leader of the Southeastern PA Chapter of the International Cesarean Awareness Network (ICAN) agrees. "Women in general are undereducated about birth, Cesareans and VBACs," she says. "So many women are told that it's not a good option, and they don't question it."
VBAC risks
While the potential risk of attempting a VBAC is low, the consequences are fairly significant, says Dr. Srinivas. The biggest risk — at .9 percent with one prior C-section and 1.4 percent with two — is a uterine rupture, which can lead to death for mom or baby.
There are complex reasons why so many women and doctors choose the risks of a surgical birth over those of a VBAC. One is that C-section risks (infection, injury to organs, problems with the placenta in future pregnancies) primarily affect the mother and not the baby, says Dr. Srinivas.
Expectant moms are finding that many doctors shy away from the risks of VBAC — and they're not entirely wrong. To provide VBAC opportunities, says Dr. Zadzielski, "You need to be able to act emergently." Hospitals must have an anesthesiologist and an obstetrician available at all times in case a repeat C-section becomes necessary, which may not be possible at smaller facilities.
"It's a risk that doctors don't seem to want to take even if the patient is willing," muses Malaga, NJ mom Jasmine Bohren. After having had two prior C-sections, 13 practitioners told Bohren they would not accept her as a VBAC candidate for the delivery of her third child. She eventually found a midwife and doctor to support her successful VBAC, in September 2013.
"They give you the worst statistics ever," she says of the doctors who focused on the potentially scary outcomes of attempting a VBAC but never mentioned the risks of a third C-section. Calabrese has noticed this phenomenon, too. "We've become desensitized to how serious a Cesarean surgery is," she notes.
Next page: VBAC candidate musts, how to find a VBAC provider & VBAC resources


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Bilstein B6 Suspension Strut AssemblyBe the first to review this product
Get Easy-to-Install Parts. No Hassle.
Improve the performance of your vehicle and seal a fade-free performance with the Bilstein B6. This suspension strut assembly is ideal for fleet vehicles, RVs or family vans.
- Monotube steering dampers
- Guarantees optimal performance
- Safe & smooth ride
- High quality
Significant Improvements to the Overall Stability of your Vehicle
The Bilstein B6 will improve the overall stability, control and handling characteristics of your vehicle. This will then give you a better security on the road.
Fade-Free Performance
The Bilstein B6 makes use of a unique, velocity sensitive digressive piston that quick reacts to changing road conditions. This provides fade-free performance.
Optimal Performance
With the Bilstein B6, you will only get optimal performance for your vehicle. And yes, you will notice the significant improvement that in your vehicle's ride quality right away.
More Comfort & Safety In the Long Run
The Bilstein B6 will deliver to you comfort and more safety in the long run. So, what are you waiting for? It's time to switch with this!
Ford (2015-2017)
Transit-150, Transit-250, Transit-350, Transit-350 HD
Jeep (2014-2017)
Cherokee, Renegade
Mercedes-Benz (2010-2017)
Sprinter 2500, Sprinter 3500
Ram (2014-2016)
ProMaster 1500, ProMaster 2500, ProMaster 3500


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Floorplan lender Northpoint Commercial Finance, which has added 25 associates in account management, credit underwriting, and sales in the past year, is looking to increase its 50 powersports OEM partnerships in 2019, according to Senior Vice President of Business Development Darren McCarthy.
McCarthy believes the relationships with current partners will spur growth at Northpoint.
"[Our dealers] may come to us for one line of credit, but they quickly realize that the service level is superior to what they may have with another lender, and they're the ones that want us," McCarthy said. "They'll promote another OEM to us, and us to the OEM, and say, 'Look, I'd like to finance this product with Northpoint.' It's organic customer references and referrals."
McCarthy said Northpoint Commercial Finance grew its net receivables 30% to 40% so far this year, to $1.3 billion. "More than anything else, we've got very, very aggressive growth goals," McCarthy said. "We are going to continue to expand and grow, and offer these powersports dealers better service at more competitive rates. We've grown our business 30% to 40% year over year, and we think we're going to maintain that kind of aggressive growth percentage." Subsequent to McCarthy's comments, the company said that it has yet to release a financial report for the year and would not confirm its financial numbers.
The Alpharetta, Ga.-based lender exceeded $1 billion in receivables last year following its August 2017 acquisition by LBC Capital — a subsidiary of Laurentian Bank of Canada. Through the acquisition, the company was able to lower its cost of capital and retain its culture and staff.


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How Much Time Do You Really Save by Speeding?
Especially during the holidays, drivers seem to be in more of a hurry to get through a longer to-do list. But will speeding really shave that much time off of the clock?
AAA recently did a study and I thought the results were kind of surprising. They looked at the time it takes to make a 30-mile trip at different speeds and this is what they found:
55 miles per hour = 32.7 minutes
65 miles per hour = 27.7 minutes (5 minutes saved)
75 miles per hour = 24 minutes (8.7 minutes saved)
This math assumes you can maintain a constant speed without slowing down for traffic, signals or curves in roads. In reality, you'd probably save only 4 minutes, at best. And keep in mind, most trips are short. The average time saved on a 5-mile trip, driving 65 mph on a 45 mph posted road, is only 1.9 minutes.
Formula: Time/mph x 60 (minutes)
Example: 30/75 = .4 x 60 = 24 minutes
Speeding is involved in about 13 percent of all crashes – and 33 percent of all fatal crashes. Speeding increases the risk of a crash, because there is less time and distance available to respond. Our reaction times-about 1 second for most drivers-don't speed up just because we are going faster.
Attention teens: Most states now have graduated driver licensing laws. That means if you have a traffic violation, you may lose your right to drive. Insurance costs could rise, and financial penalties could be steep. Is possibly saving a few minutes on the road really worth the risk?
Bottom line? Slow down and stay safe this holiday season


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Chris Keenist, is a proud graduate of Oxford highschool, where he played football and basketball. Last September 4th he was on the field with his Adrian college teammates as they opened the 2011 football season. Less than two weeks later he was rushed to Beaumont Hospital where he was diagnosed with Cardio Myopathy, but he was one of the lucky ones. The condition was discovered before it was too late. Many young athletes aren't so lucky.
Over the last eight years, at least 29 Michigan highschool students have died because of sudden cardiac arrest. The majority of these young athletes who died suddenly have no symptoms and most of the victims have had Cardio Myopathy, which is one of the most serious heart problems facing young athletes today.
Cardio Myopathy can be diagnosed in a pre-participation screening which includes a brief heart exam, a twelve-lead electrocardiogram and, if necessary, an echocardiogram.
Help CBS Radio Detroit save young lives by supporting the Student Heart Check program. This program has already helped detect some serious heart issues among our young athletes and undoubtedly saved lives.
With your support, we'll save more lives. The next young athlete saved may be someone you know and love.
$10 can save a life! Text "SCREEN" to 50555 to donate $10.
My wife and I decided to have our 14 y/o son screened due to the reports of the sudden death of the high school basketball player earlier this year. Our son, like him, is very athletic and has participated in organized sports since age 8. He excelled in baseball, and was looking forward to participating in high school baseball until a heart screening identified that he has hypertrophic cardiomyopathy (HCM). Needless to say we were floored, but very thankful that we found out like this and before it was too late.
If you have young kids that are active, please take advantage of this service in your area. My son is very fit, and I otherwise would have no clue by looking at him that anything was wrong.


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One timber frame home brings barn-style beauty to the Long Island Sound.
Ken crafted the 8-foot-long American flag from driftwood he found on the beach
Reclaimed brick floors in a herringbone pattern strike a rustic note in the kitchen. Neutral cabinets and countertops add to the home's farmhouse feel.
The home's oak wood floors, showcased here in the great room, are not dimensional lumber, but engineered. "The engineered lumber is more stable for conducting the radiant heat that we installed beneath the floors," Ken explains.
The home's double-sided, see-through fireplace is faced with the same reclaimed brick that's found in the entryway and kitchen. Unique light fixtures bring a modern, artistic flair to the home's interior living spaces.
It's such a beautiful place. It's the perfect combination of traditional and modern." – homeowner, Ken Zahler
The traditional clawfoot tub fits in perfectly in the first-floor master bathroom.
"All of the shutters were custom made within a quarter of an inch of the windows, so they're a perfect fit," says Ken.
Sliding doors and shutters (an authentic barn feature) were installed in many of the doorways and windows on the second floor
Painted in a natural gray hue, the home perfectly complements the surrounding landscape. A hand-split cedar shingled roof tops off the design.
Our Favorite Feel-Good Timber Home Stories
A Wisconsin Timber Home With Its Own Private Ga...
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This Tennessee Home Is Cozy, But Far From Conve...
A Sleek and Modern Timber Home in Nebraska


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At Village of Moms, you can find a community of mothers who have experience with raising children. Everyone here has come together to share their advice, stories, and tips on coping with the various aspects of motherhood. You don't need to feel alone when you're trying to deal with your kids – we are always here to help you.
Whether you want breastfeeding tips, inspirational quotes, or help dealing with loss, Village of Moms is where you'll find the support you need. Share a resource, link, experience or encouragement inspiring story that you think might help support other moms going through this to let them know they are not alone.
If you need to reach out for help or would like to share your own personal experiences, please don't hesitate to contact us using the form provided on this page.
Contact Information
North Vancouver, BC


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Professional Security Camera Installation Services Junction N15
Need help in upgrading your home video surveillance system? Locksmith Earl is a professional security company which offers different and efficient surveillance solutions in Junction. All our CCTV installers are vetted, experienced and trained to work with all kinds of modern CCTV system security equipment. We offer various CCTV related services, including supply, maintenance, installation and repairs for commercial and residential properties.
We guarantee that:
- Our services and products come with monitoring, plus access to the surveillance data via 24/7
- We use the latest equipment with smartphone video quality which is court-accepted
- We can provide exceptional of your current CCTV emergency repair and regular maintenance
- Our systems are systems and cameras professionally with minimum blind spots
To get more , or to information a service, contact our customer support operators right away. 24/7.
Why locals prefer our CCTV services
Our surveys show that we are a top preferred local CCTV company in Junction. This is due to fact that we offer the most cost-effective CCTV installation prices in the area. Our CCTV supply, installation and maintenance are easy and fast. We offer the latest solutions and security camera (CCTV) installation, fitting and repair in the region. Apart from CCTV installation, we provide CCTV intruder alarms and same day CCTV services and repairs of home and business CCTV surveillance systems. We also offer remote access CCTV monitoring via your smartphone, computer or other mobile devices.
If you need one or more of these services and you are in Junction, call us right away!
The cameras we work with
The security camera equipment which we use for our CCTV systems includes:
- Motion detection camera, Home security cameras, Outdoor security camera
- Night vision & day cameras, Infrared security cameras, Dome security cameras
- Monochrome cameras, Bullet security cameras, Covert cameras
- Wide dynamic range (HDR) cameras, Pan tilt zoom (PTZ) cameras
- Dummy cameras, Samsung cameras, Dahua cameras
- Wireless security cameras, Internet security cameras, Pelco cameras
- IP security cameras, Remote access CCTV monitoring
- Auto-zoom security cameras, Network cameras, High-definition (HD) cameras
We have many customers in the area
If you need reliable CCTV home and business maintenance, repairs or installers in Junction, contact us.
- Police forces, Airports, Industrial estates
- Building contractors, Retail outlets, Construction companies
- Private residential homes, Healthcare facilities
- Government buildings, Education estates, Clubs
So, if you are in need of a expert CCTV professional service in the area of Junction, go ahead and book it!
More information on our company in Junction
As part of our security related services, we offer expert CCTV installation services in Junction. We have been installing, maintaining and repairing the CCTV camera security systems in the homes and business properties of our customers in the area ever since 2004.
Our engineers are all police and NIS vetted. They have the knowledge to work with all kinds of surveillance cameras. We offer a professional and quality guarantee services at the most affordable CCTV installation prices in the area.
We are considered to be one of the most reliable companies for CCTV supply, installation and maintenance in Junction.
How to book our CCTV services in Junction N15
To book our installation, repair or maintenance services for your CCTV surveillance system, contact us on 020 3404 1580 or online via the booking form. We offer same day booking and emergency booking for urgent cases with a response time of 30 minutes. There is no deposit for booking.


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Find, identify and correct the errors in the following examples.
" 'Sadly, these guys are not alone, this is not a unique case,' said Baeumler, whose Disaster DIY and Leave it to Bryan series on the Home & Garden Television Network are filled with similar stories of home renos gone horribly wrong."
Doug Schmidt, "TV star leads extreme makeover at family's mould-infested home", The Windsor Star, Thursday, October 10, 2013.
"Actually, I'm kind of offended with the fact that they want to put calories on a menu," Glenn said. "I could care less that they want to put calories on a menu when the country doesn't even take the initiative to tell me where my food comes from."
Glenn Swinton, in a story by Beatrice Fantoni, "It's coming: calories on menus", The Windsor Star, Thursday, October 10, 2013.
The word for today is "rhetorical".
What part of speech is "rhetorical"?
Define "rhetorical" and use it in a sentence that demonstrates its meaning.
Corrections and explanations for this week's entries will be posted tomorrow, Friday.
BONUS: Read the sentence above and identify the designation for the word "Friday". Explain it correctly and receive a GOLD STAR.


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Beautiful Concertante model cello hand finished by Paul Stanton
Stradivari pattern cello. "Our Concertante model" from the workshop of Paul Michael Stanton. These instruments are entirely hand made by European trained violin makers. The front is carved from high quality evenly spaced tonewood. The two piece back, ribs and scroll from highly figured selected maple. The instrument has an excellent tone and is evenly balanced over the four strings. The cello is covered with a deep chestnut antique varnish over a strong golden ground. Please note that unlike many sellers I am a professional violin maker and Newark violin school graduate and each instrument is set up to the highest of professional standards before it leaves my workshop. The ebony pegs complete with Parisian eye detail are re-seated to ensure smooth operation. The finger board is re-surfaced and an Aubert bridge is hand cut to ensure correct string heights (I normally charge £95 for this alone). In addition I lower the nut to the correct tolerences to enable extending back into the half positions as trouble free as possible. The cello comes with a "Wittner" integrated tailpiece for ease of tuning and Jarger strings to bring out the very best in the tone of the instrument. Altogether a superb looking cello that we don't think you will better for less than £5000. Suitable for players of all levels. Complete with good quality bow, styro foam hard case with music pocket and provision for two bows, rosin and carry straps.


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Contoured Comfort
Comfortable, deep & spacious, each seat & lounger on the Prince Pro hot tub is structured to create the complete spa experience.
Contoured lines merge both body & surface together, with the added depth that Prince Pro offers - the highest quality of relaxation is achieved.
All Prince Pro models are 92cm deep & seats measure between 50 and 55cm wide.


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We've reported previous about LED nails – http://ow.ly/ukuv30eTqsu – which didn't quite get the approval from you guys. However, LED eyelashes are here and they do look a lot better! These LED lashes are applied to the eyelid with adhesive lash glue, and the cheeky connector is hidden behind the ear. They even come with different settings (think Christmas tree lighting) for whatever you're in the mood for!
What do you make of these?
Related Articles:


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In the six years since Nassau County froze property assessments, there have been 800,775 grievances filed by homeowners. But there are only 386,101 parcels eligible for such protests. It's a broken system: No one can afford to forgo fighting an assessment repeatedly, and everyone loses out except the tax-reduction companies many homeowners pay to generate savings that in the long run don't exist.
Successful tax grievances do not lower the tax burden. They lower the assessed values of properties, which means a higher rate needs to be charged on all property to make up the difference.
About 75 percent of residential grievances succeed in Nassau, and each lowers the assessed value of property in its taxing districts. For the past two years, the average decrease in assessment won was 8 percent. Since 2010, Nassau's total taxable assessed valuation has gone down 28 percent. But the value of residential property has increased 12 percent.
The assessments are diverging from reality. So tax rates on the shrinking base are expanding.
The average school district tax-rate increase in Nassau in 2016 was 4.86 percent, but the average tax levy increase was just 1.69 percent. In Long Beach, the rate increase was 8.21 percent, but the levy increase was just 3.19 percent. In Rockville Centre, the rate increased 8.13 percent, but the levy increased just 2.81 percent. If you do not grieve and your tax rate goes up 8 percent, so do your taxes. Someone who gets an 8 percent reduction in assessed value and faces an 8 percent rate increase would pay no more in taxes that year, but also have no savings.
Anyone who didn't win a grievance suffered a huge hike in their taxes. But anyone who won a grievance didn't get the savings he or she might have expected. Most everyone is filing grievances and most everyone's rate is going up over time as assessments go down. And millions of dollars are going to tax-appeal companies, which get a piece of what they say they've saved clients.
County Executive Edward Mangano is hamstrung by a county-guarantee law that says Nassau must refund all tax overpayments created by successful assessment appeals, even though 84 percent of the overpayments go to other taxing entities. So he now settles almost all residential appeals before the tax roll is set. That has eliminated $20 million a year in refunds, but the county has smoothed the process by lowering the assessments of homeowners even if properties are fairly valued.
Several law changes are needed in Albany to fix the problem, which exists across the state, although not as severely as in Nassau:
- Property owners who grieve should face the risk of having their assessments increased, as in New Jersey and Connecticut.
- State law should create a margin of error below which owners get no rebate. In New Jersey, a valuation must be off 15 percent before a court can change it. In New York, any discrepancy creates a reduction.
- The county guarantee, unique to Nassau, should be repealed.
And, of course, Nassau should regularly and accurately assess properties, something it has never been much good at.
This system is a disaster, with no winners except tax-grievance companies.— The editorial board