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Regulation of Flowering Time and Floral Organ Identity by a MicroRNA and Its APETALA2-Like Target Genes
Milo J. Aukerman, Hajime Sakai
Milo J. Aukerman
E. I duPont de Nemours and Company, Agriculture & Nutrition, Newark, Delaware 19711
Hajime Sakai
Published November 2003. DOI: https://doi.org/10.1105/tpc.016238
Corrections - February 01, 2004
MicroRNAs (miRNAs) are ∼21-nucleotide noncoding RNAs that have been identified in both animals and plants. Although in animals there is direct evidence implicating particular miRNAs in the control of developmental timing, to date it is not known whether plant miRNAs also play a role in regulating temporal transitions. Through an activation-tagging approach, we demonstrate that miRNA 172 (miR172) causes early flowering and disrupts the specification of floral organ identity when overexpressed in Arabidopsis. miR172 normally is expressed in a temporal manner, consistent with its proposed role in flowering time control. The regulatory target of miR172 is a subfamily of APETALA2 (AP2) transcription factor genes. We present evidence that miR172 downregulates these target genes by a translational mechanism rather than by RNA cleavage. Gain-of-function and loss-of-function analyses indicate that two of the AP2-like target genes normally act as floral repressors, supporting the notion that miR172 regulates flowering time by downregulating AP2-like target genes.
MicroRNAs (miRNAs) are noncoding RNAs of ∼21 nucleotides in length that have been identified in both animals and plants (Lagos-Quintana et al., 2001, 2002; Lau et al., 2001; Lee and Ambros, 2001; Llave et al., 2002a; Mourelatos et al., 2002; Park et al., 2002; Reinhart et al., 2002). They are processed from longer precursor transcripts that range in length from ∼70 to 200 nucleotides, and these precursor transcripts have the ability to form stable hairpin structures. In animals, the enzyme involved in processing miRNA precursors is called Dicer, an RNase III–like protein (Grishok et al., 2001; Hutvagner et al., 2001; Ketting et al., 2001). Plants also have a Dicer-like enzyme encoded by DICER-LIKE1 (Schauer et al., 2002), and recent evidence indicates that it, like Dicer from animals, is involved in processing the hairpin precursors to generate mature miRNAs (Park et al., 2002; Reinhart et al., 2002). Furthermore, it is becoming clear from recent work that at least some miRNA hairpin precursors originate as longer polyadenylated transcripts, and several different miRNAs and associated hairpins can be present in a single transcript (Lagos-Quintana et al., 2001; Lee et al., 2002).
In animals, there is direct evidence indicating a role for specific miRNAs in development. The lineage (lin)-4 and lethal (let)-7 miRNAs in the worm Caenorhabditis elegans have been found to control temporal development, based on the phenotypes generated when the genes producing the lin-4 and let-7 miRNAs are mutated (Lee et al., 1993; Reinhart et al., 2000). In addition, both miRNAs display a temporal expression pattern consistent with their roles in developmental timing. Other animal miRNAs display developmentally regulated patterns of expression, both temporal and tissue specific (Lagos-Quintana et al., 2001, 2002; Lau et al., 2001; Lee and Ambros, 2001), leading to the hypothesis that miRNAs, in many cases, may be involved in the regulation of important developmental processes. Recent studies in Drosophila and mammals have associated specific developmental phenotypes with reduced expression of specific miRNAs (Brennecke et al., 2003; Xu et al., 2003), providing support for the proposed developmental role of miRNAs. In plants, the differential expression patterns of many miRNAs also suggest a role in development (Llave et al., 2002a; Park et al., 2002; Reinhart et al., 2002). Not much is known, however, about the functioning of plant miRNAs in specific developmental processes, because phenotypes associated with the overexpression or underexpression of specific miRNAs have not been found.
miRNAs appear to regulate target genes by binding to complementary sequences located in the transcripts produced by these genes. In lin-4 and let-7, the target sites are located in the 3′ untranslated regions of the target mRNAs (Lee et al., 1993; Wightman et al., 1993; Reinhart et al., 2000; Slack et al., 2000). Binding of the lin-4 or let-7 miRNA appears to cause the downregulation of steady state levels of the protein encoded by the target mRNA without affecting the transcript itself (Olsen and Ambros, 1999). On the other hand, recent evidence suggests that in some cases miRNAs can cause specific RNA cleavage of the target transcript within the target site (Hutvagner and Zamore, 2002; Llave et al., 2002b). miRNAs entering the RNA cleavage pathway are analogous to the 21- to 25-nucleotide short interfering RNAs generated during RNA interference in animals and post-transcriptional gene silencing in plants (reviewed by Pickford and Cogoni, 2003) and likely are incorporated into an RNA-induced silencing complex that is similar or identical to that seen for RNA interference.
It has been suggested that the mode of regulation used by a particular miRNA (i.e., either translational regulation or RNA cleavage) may be determined by the extent of sequence complementarity between the miRNA and its target (Rhoades et al., 2002). miRNAs that have a significant number of mismatches and bulges when aligned to their targets are predicted to cause translational regulation, as in the cases of lin-4 and let-7. On the other hand, a miRNA with perfect or nearly perfect complementarity with its target is predicted to cause RNA cleavage, and recent studies in both plants and animals have provided support for this idea (Hutvagner and Zamore, 2002; Llave et al., 2002b; Tang et al., 2003). Plant miRNAs share perfect or nearly perfect complementarity with their targets (Rhoades et al., 2002), which implies that many of the plant miRNAs regulate their targets by an RNA cleavage mechanism.
One striking observation about the putative target transcripts of plant miRNAs is that most of them encode members of transcription factor families that have been implicated in plant developmental patterning or cell differentiation (Rhoades et al., 2002). Plant miRNAs thus are likely to control developmental decisions by downregulating important developmental transcription factors. To date, however, there are limited data directly demonstrating a role for miRNAs in plant development. Llave et al. (2002b) have shown that a transcript for a SCARECROW-like transcription factor is a target of the Arabidopsis miRNA miR171; however, these studies were performed in a heterologous species, and no plant phenotype associated with miR171 was reported. Another miRNA, miR165/166, is predicted to target the homeodomain/Leu zipper transcription factor genes PHABULOSA (PHB) and PHAVOLUTA (PHV) (Rhoades et al., 2002), and particular mutant alleles of these genes contain single-base mutations within the miR165/166 target site. In these alleles, the PHB and PHV gene products are expressed ectopically (McConnell et al., 2001), suggesting that miR165/166 normally limits the domain of PHB and PHV expression, and RNA cleavage studies have demonstrated that PHB and PHV are in fact targets of miR165/166 (Tang et al., 2003). Nonetheless, mutants of miR165/166 have not been reported; therefore, the true function of miR165/166 in plant development can only be inferred by the phb and phv mutant phenotypes.
Using a forward-genetics approach, we have identified a miRNA that confers a developmental phenotype in Arabidopsis. This miRNA, miR172 (Park et al., 2002), causes early flowering and defects in floral organ identity when overexpressed. The predicted target of miR172 is a small subfamily of APETALA2-like transcription factors (Okamuro et al., 1997), including APETALA2 (AP2) itself. Surprisingly, miR172 appears to downregulate the AP2-like target genes via a translational mechanism rather than by RNA cleavage, even though it shares nearly perfect complementarity with these targets. In a recent report, Chen (2003) used a reverse-genetics approach to characterize miR172. Whereas that study focused primarily on miR172 regulation of AP2, we have extended the analysis to other AP2-like targets of miR172. Specifically, we have determined that overexpression of one of the AP2-like target genes, TARGET OF EAT1 (TOE1), causes late flowering. This result, in conjunction with loss-of-function analyses of TOE1 and another target gene, TOE2, indicates that at least some of the AP2-like genes targeted by miR172 normally function as floral repressors. Finally, we present a model that postulates that downregulation of AP2-like target genes by miR172 during early wild-type seedling development relieves floral repression by these genes, resulting in the promotion of flowering.
Overexpression of EARLY ACTIVATION TAGGED Causes Early Flowering and an ap2 Phenotype
Flowering time in Arabidopsis is controlled by several environmental and endogenous pathways, and >80 genes participate in regulation of the floral transition in this species (Simpson and Dean, 2002). In an attempt to identify novel genes involved in flowering time control, we screened an activation-tagging population (Weigel et al., 2000) (see Methods) for mutants displaying altered flowering time. Several early- and late-flowering lines were identified, and here we describe two of these mutants, early activation tagged, dominant (eat-D) and toe1-1D (see below). eat-D flowers extremely early, as indicated by visual inspection (Figure 1B) and by measuring rosette leaf number (Table 1). In addition, EAT-D displays floral defects (Figure 1D) that are virtually identical to those observed for strong ap2 mutant alleles (Bowman et al., 1991), including the complete absence of petals and the transformation of sepals to carpels.
EAT Overexpression Phenotype.
(A) Wild-type (Columbia [Col-0] ecotype) plant, 3.5 weeks old.
(B) eat-D plant, 3.5 weeks old.
(C) Wild-type flower.
(D) eat-D flower. Note the absence of second-whorl organs (petals). The arrow indicates a sepal with ovules along the margins and stigmatic papillae at the tip. The eat-D phenotype is virtually identical to that of strong ap2 mutant alleles.
(E) Cauline leaf margin from a 35S-EAT plant. Arrows indicate bundles of stigmatic papillae projecting from the margin.
(F) Solitary gynoecium (arrow) emerging from the axil of a cauline leaf of a 35S-EAT plant.
Flowering Times and Floral Phenotypes of Lines Used in This Study
We mapped the activation-tagged T-DNA insert in eat-D to chromosome 5, between the annotated genes At5g04270 and At5g04280 (Figure 2A). We then used 5′ and 3′ rapid amplification of cDNA ends (RACE) PCR with primers located within this region to identify a 1.4-kb transcript, which we named EAT, that is upregulated in eat-D (Figure 2A; see also Figure 4A). When the 1.4-kb EAT cDNA was fused to the constitutive 35S promoter of Cauliflower mosaic virus and the resultant 35S-EAT construct was introduced into wild type Columbia (Col-0) plants by Agrobacterium tumefaciens–mediated transformation (Clough and Bent, 1998), the 35S-EAT transformants displayed the identical early-flowering and ap2-like phenotypes seen in EAT-D (Table 1, Figures 1B and 1D). 35S-EAT plants had a normal response to photoperiod, flowering later under short days than under long days (Table 1). Many of the 35S-EAT transformants occasionally displayed floral defects in addition to those seen in eat-D, including stigmatic papillae on cauline leaf margins (Figure 1E) and the formation of a complete or partial flower rather than a secondary inflorescence in the axils of cauline leaves (Figure 1F). Therefore, ectopic expression of EAT in 35S-EAT plants affects both flowering time and the specification of floral organ identity.
EAT Contains a miRNA That Is Complementary to AP2.
(A) Location of EAT on chromosome 5. The T-DNA insertion and the orientation of the 35S enhancers are indicated. The 21-nucleotide sequence corresponding to miR172a-2 is shown below the EAT gene.
(B) Structure of the EAT precursor transcript. The polyadenylated and 5′ capped, unspliced transcript is shown, along with the positions of two introns. Two alternatively spliced transcripts, one with both introns removed and the other with intron 2 spliced out, also accumulate in wild-type plants. The location of the miR172a-2 miRNA is indicated with an asterisk.
(C) The putative 21-nucleotide miR172a-2/AP2 RNA duplex is shown below the gene structure of AP2. Two mismatches are indicated in boldface type. The boxed area delineates the coding region (ATG to stop) of the AP2 transcript, and the black boxes show the locations of the two AP2 domains.
(D) Alignment of the AP2 21-nucleotide target site (black bar at top) and surrounding sequence with three other Arabidopsis AP2 family members and two maize AP2 genes (IDS1 and GL15). Nucleotide positions of the sequences within each cDNA sequence are indicated at right. Black boxes indicate nucleotides that are highly similar to other family members, and gray boxes show nucleotides that are less well conserved.
(E) Alignment of miR172a-2 miRNA (black bar at top) and surrounding sequence with other miR172 sequences from Arabidopsis (miR172a-1, miR172b-1, miR172b-2, and miR172c), tomato (AI484737), soybean (BI320499), potato (BQ114970), and rice (AP003277, AP004048, AP005247, and ctg7420). Black and gray boxes are as described for (D).
The EAT Transcript Is the Precursor RNA for a Member of the miR172 Family
EAT produces a 1417-nucleotide noncoding RNA that is predicted to be 5′ capped and polyadenylated (Figure 2B), based on our RACE-PCR methodology (see Methods). We also identified two splice variants of EAT: a short form in which two introns are removed, and an intermediate form in which only one intron (intron 2) is spliced out. All three transcripts are present at similar levels in wild-type plants (M.J. Aukerman, unpublished results). Basic Local Alignment Search Tool (BLASTN and BLASTX) searches of several databases with the 1.4-kb EAT cDNA revealed no extensive nucleotide or predicted amino acid sequence identity between EAT and any other gene. However, we did identify a 21-nucleotide stretch in the middle of the EAT transcript that is identical to miR172a-2 (Figure 2A), a recently identified miRNA (Park et al., 2002). Both splice variants of EAT also contain this 21-nucleotide sequence (Figure 2B, asterisk). To confirm the functional importance of the miR172a-2 sequence within the EAT cDNA, we generated a mutant form of EAT in which the miR172a-2 sequence was deleted and made a construct consisting of this mutant EAT cDNA, eatdel, driven by the 35S promoter. Transgenic plants carrying this 35S-eatdel construct flowered with the same number of leaves as the wild type and had normal flowers (Table 1), indicating that the miR172a-2 sequence is necessary to confer both the flowering-time and floral organ identity phenotypes seen in EAT-overexpressing lines.
As noted by Park et al. (2002), the 21-nucleotide miR172 miRNA has the potential to form an RNA duplex with a sequence near the 3′ end of the coding region of AP2 (Figure 2C). It is likely, then, that overexpression of miR172a-2 causes the observed ap2 phenotype by binding to this target site in AP2, leading to a reduction in AP2 expression (see below). Similar to other plant miRNAs, miR172 shares nearly perfect complementarity with its target (19 of 21 nucleotides). The target site for miR172 is found in a similar location in three other Arabidopsis AP2 family members that we have named TOE1, TOE2, and TOE3 (corresponding to At2g28550, At5g60120, and At5g67180, respectively) and in the maize AP2 genes INDETERMINATE SPIKELET1 (Chuck et al., 1998) and GLOSSY15 (Moose and Sisco, 1996) (Figure 2D). We also found the miR172 target site in AP2 family members from many other plant species, including soybean, rice, wheat, tomato, and pea (M.J. Aukerman, unpublished results). The conservation of this 21-nucleotide sequence in a subset of AP2 genes from both monocots and dicots suggests that this sequence is of functional significance and is likely a cis-acting factor that regulates the expression of these particular AP2 family members in response to miR172-like miRNAs.
There is an additional copy of the miR172a-2 miRNA in the Arabidopsis genome on chromosome 2 (miR172a-1), and miR172a-2 is highly similar to three other Arabidopsis loci (Figure 2E). Like the miR172a-2 miRNA, all four reiterations of the sequence are in intergenic regions (i.e., between the Arabidopsis genes currently annotated in GenBank). In addition, the sequence was found in ESTs from tomato, potato, and soybean, and four copies were found in the genomic sequence of rice (Figure 2E). To determine whether another member of the miR172 family, miR172a-1, would confer a phenotype similar to that of miR172a-2, we generated a construct containing the 35S promoter fused to the genomic region surrounding miR172a-1. Plants containing the 35S-miR172a-1 construct flowered early and displayed an ap2 phenotype (Table 1), indicating that miR172a-1 behaves in an identical manner to miR172a-2 when overexpressed.
Expression Analysis of miR172
RNA gel blot analysis using a probe antisense to the miR172a-2 miRNA identified a small single-stranded RNA of 21 to 25 nucleotides accumulating to much higher levels in eat-D plants relative to the wild type (Figure 3A). The small amount of transcript seen in the wild type presumably represents endogenous levels of not only the miR172a-2 miRNA but also its family members, which are similar enough to cross-hybridize with the probe. The predicted miR172a-2 hairpin precursor is 95 nucleotides in length (Park et al., 2002), and we detected a transcript of approximately that size accumulating in eat-D that likely represents unprocessed miR172a-2 hairpin precursor (Figure 3A, arrowhead). S1 nuclease mapping of the miR172a-2 miRNA (Figure 3B) provided independent confirmation of the 5′ end of miR172 reported by Park et al. (2002).
miR172 Expression.
(A) RNA gel blot of 50 μg of total RNA from wild-type Col-0 (lanes 3 and 7) and eat-D (lanes 4 and 8). RNA was isolated from the aboveground parts of plants that had already bolted. Blots were probed with sense (lanes 1 to 4) or antisense (lanes 5 to 8) oligonucleotides to miR172a-2 miRNA. One hundred picograms of sense oligonucleotide (lanes 2 and 6) or antisense oligonucleotide (lanes 1 and 5) was loaded as a hybridization control. Nucleotide length markers are indicated at left. The arrowhead shows the putative hairpin precursor of miR172a-2. Ethidium bromide–stained 5S RNA served as a loading control for lanes 3, 4, 7, and 8.
(B) S1 nuclease mapping of miR172a-2 miRNA. A 5′ end labeled probe was undigested (lane 1) or digested after hybridization to total RNA from wild-type (lane 2), eat-D (lane 3), or tRNA (lane 4) plants. RNA samples in lanes 2 and 3 were isolated from aboveground parts of plants that had already bolted. Nucleotide length markers are indicated at left.
(C) RNA gel blot of 30 μg of total RNA from wild-type Col-0 (Col), luminidependens (ld), or constans (co) probed with antisense oligonucleotide to miR172. Plants were harvested at 16 days after germination. 5S RNA, loading control.
(D) RNA gel blot analysis of 30 μg of total RNA from wild-type seedlings harvested at 2, 5, 9, 12, and 16 days after germination or from mature leaves (L) and floral buds (F). Probes for miR172 and 5S RNA are indicated at left.
(E) Quantification of miR172 transcript abundance (normalized to 5S RNA) with respect to developmental time (i.e., days after germination). The normalized miR172 RNA level at 16 days after germination was set arbitrarily to 1.
The expression of miR172 was regulated temporally, with no miR172 transcripts detected 2 days after germination, and progressively more steady state transcript accumulation was seen as the plant approached flowering (Figures 3D and 3E). The miR172 transcripts also accumulated after flowering had occurred in both leaves and floral buds. We assessed the levels of miR172 in two mutants, luminidependens and constans, and found that miR172 expression was not altered in either of these mutants (Figure 3C), indicating that miR172 is not found downstream of these genes. In addition, the levels of the miR172a-2 precursor transcript were identical in plants grown in long days versus those grown in short days (M.J. Aukerman, unpublished results).
miR172 Is a Translational Repressor of Its AP2-Like Target Genes
Immunoblot analysis with an antibody specific for AP2 (see Methods) indicated that the AP2 protein was reduced dramatically in the miR172a-2 overexpression (eat-D) line relative to the wild type, whereas the AP2 transcript accumulated to normal levels in eat-D (Figure 4A). Likewise, mRNA levels for the AP2-like target genes TOE1 and TOE2 were not reduced in the eat-D line. These data suggest that the miR172a-2 miRNA negatively regulates AP2 (and likely its other targets as well) at the level of translation and not by RNA cleavage. This result was unexpected in light of the report from Kasschau et al. (2003), in which cleavage products derived from the miR172 target sites in the AP2-like genes were identified. We used the same reverse transcriptase–mediated (RT) PCR procedure described by those authors and confirmed that cleavage products can be detected for the AP2-like target genes in both wild-type and eat-D plants (Figure 4B). Nevertheless, we detected no cleavage products of the appropriate size on RNA gel blots (Figure 4A), even when miR172 was overexpressed.
miR172 Downregulates AP2.
(A) At top and bottom left are RNA gel blots of 1 μg of poly(A+) RNA from the wild type (wt) and eat-D. RNA from each genotype was isolated from floral buds. The blots were hybridized with probes specific for the AP2-like genes indicated below or at left. At bottom right are immunoblots of proteins from wild-type (wt) and eat-D floral buds probed with AP2 antibody. AP2 is severely reduced in eat-D. RbcL, large subunit of ribulose-1,5-bisphosphate carboxylase as a loading control.
(B) Target sites for miR172 in three AP2-like transcripts, with the 5′ ends of RNA cleavage products that were identified by 5′ RACE PCR indicated by arrows. The numbers of 5′ RACE clones sequenced that correspond to each cleavage product are indicated.
For each AP2-like gene, a gene-specific probe that recognizes sequences 3′ to the miR172 target site was used for hybridization; therefore, we expected to detect 3′ cleavage products in the region of the gel corresponding to fragment lengths of 200 to 500 nucleotides (e.g., the predicted AP2 cleavage product is ∼300 nucleotides in length). Cleavage products corresponding to the 5′ end of each transcript would not be detected, because the RNA samples were poly(A+) selected. Because we did not detect miR172-directed 3′ cleavage products by RNA gel blot analysis, the cleavage products detected by PCR likely represent a very small fraction of the total transcript population produced from the AP2-like genes. This notion is supported by the fact that overexpression of miR172 did not change the steady state levels of the AP2-like target genes (Figure 4A). We conclude that miR172 regulates its target genes by a translational mechanism and that the small amount of RNA cleavage observed likely is the result of overlap between the translational and RNA cleavage pathways (see Discussion for more details).
Two AP2-Like Targets of miR172 That Function as Floral Repressors
In the same genetic screen that identified the early-flowering eat-D mutant, we identified an activation-tagged late-flowering mutant called toe1-1D. toe1-1D displayed no additional phenotypes besides late flowering (Table 1), and the late-flowering phenotype cosegregated with a single T-DNA insertion (M.J. Aukerman, unpublished results). Sequence analysis of the T-DNA insert in toe1-1D indicated that the 4× 35S enhancer was located ∼5 kb upstream of TOE1 (Figure 5A), one of the AP2-like target genes that contains a target site for miR172a-2 (Figure 2D). Semiquantitative RT-PCR analysis using primers specific for TOE1 indicated that the transcript corresponding to this gene was expressed at 23-fold higher levels in toe1-1D relative to the wild type (Figure 5B). To confirm that overexpression of TOE1 causes late flowering, we fused a genomic region containing the entire TOE1 coding region to the 35S promoter and created transgenic plants containing this construct. Transgenic 35S-TOE1 plants flowered much later than wild-type plants and also later than toe1-1D (Table 1). This late-flowering phenotype was observed in multiple independent transformants (M.J. Aukerman, unpublished results).
Activation-Tagged and Loss-of-Function Alleles of AP2-Like Target Genes.
(A) Location of the T-DNA insert in toe1-1D. The 35S enhancers are located ∼5 kb upstream of TOE1 (At2g28550).
(B) Semiquantitative RT-PCR analysis of TOE1 and ACT11 expression in the wild type versus toe1-1D. RNA from each genotype was isolated from floral buds. Inclusion of reverse transcriptase (RT) is indicated by + or − above the gels.
(C) eat-D suppresses the phenotype of toe1-1D. Flowering-time phenotypes (expressed as rosette leaf number) of F1 plants derived from the cross of a toe1-1D heterozygote to an eat-D homozygote. Approximately 50% of the F1 plants contained the toe1-1D allele, based on PCR genotyping; these plants were early flowering, indicating the suppression of toe1-1D (which overexpresses TOE1) by eat-D (which overexpresses miR172). In this experiment, wild-type control plants flowered with ∼11 leaves.
(D) Locations of the T-DNA inserts in toe1-2 and toe2-1. Exons are indicated by solid bars, and the target sequences for miR172 are indicated with asterisks. Brackets delineate the two AP2 domains contained in each gene.
To assess the ability of miR172a-2 to regulate TOE1, a plant homozygous for eat-D (and thus overexpressing miR172a-2) was crossed to a plant heterozygous for toe1-1D (and thus overexpressing TOE1). In this cross, all of the F1 progeny were expected to contain one copy of eat-D and 50% of the F1 progeny also should have had one copy of toe1-1D. F1 progeny were scored for the presence or absence of the toe1-1D allele by PCR and also were scored for flowering time. All of the F1 plants were early flowering regardless of whether or not they contained a copy of the toe1-1D allele (Figure 5C), indicating that overexpression of miR172a-2 can suppress the effects of TOE1 overexpression. This result is consistent with a model in which miR172a-2 directly downregulates TOE1 by binding to its cognate target site. We observed late-flowering toe1-1D segregants in the subsequent generation (M.J. Aukerman, unpublished results), which excludes the possibility that early flowering in the F1 generation was attributable to cosuppression occurring at the TOE1 locus.
To determine the effects of reducing TOE1 function, we identified plants containing a T-DNA insertion in TOE1 (Figure 5D). In addition, we identified a T-DNA mutant for TOE2, a closely related AP2-like gene that also contains the miR172a-2 target sequence. Given the locations of the inserts either upstream of (toe2-1) or within (toe1-2) the highly conserved AP2 domain, it is likely that both mutations result in the complete loss of function of the respective gene. We determined that plants homozygous for the toe1-2 T-DNA allele were slightly early flowering relative to the wild type, whereas toe2-1 plants were not significantly early flowering (Table 1). The two mutants were crossed, and the double mutant was identified by PCR genotyping. The toe1-2 toe2-1 double mutant was earlier flowering than either individual mutant (Table 1), suggesting that the genes have overlapping function. The early-flowering phenotype of toe1-2 toe2-1 is consistent with a model in which miR172 causes early flowering by downregulating several AP2-like floral repressors, including TOE1 and TOE2. Interestingly, toe1-2 toe2-1 is not as early flowering as miR172-overexpressing lines (cf. 35S-EAT; Table 1), which suggests that other AP2-like targets of miR172a-2 (e.g., AP2 itself or TOE3) also act as floral repressors. Because ap2 single mutants are not early flowering (Bowman et al., 1991), any potential negative regulation of flowering by AP2 must be normally masked by genetic redundancy.
Using an activation-tagging approach, we have identified a miRNA involved in the control of flowering time and floral organ identity. Overexpression of EAT, which produces a precursor transcript for miR172a-2, causes extremely early flowering and floral organ identity defects. The predicted target of miR172 is a small subfamily of AP2-like genes, including AP2 itself, and higher levels of miR172 are associated with the downregulation of AP2 gene expression at the translational level. In a recent report, miR172 was characterized by a reverse-genetics approach (Chen, 2003), and our findings support the general conclusions of that work. Whereas Chen focused primarily on miR172 regulation of AP2 and its effects on floral organ identity, we have found that two of the other AP2-like targets of miR172, TOE1 and TOE2, function in the control of flowering time as floral repressors. Because the target site sequences of these other AP2-like target genes are virtually identical to that of AP2, it is highly likely that they also are downregulated by miR172, which would explain the early-flowering phenotype associated with miR172 overexpression.
Based on the overexpression phenotypes of both miR172a-1 and miR172a-2 (Table 1), one would expect that loss-of-function alleles of these miRNAs would have the opposite phenotype. This prediction may be difficult to test, however, because of the high degree of sequence similarity among the predicted mature miR172 miRNAs (Figure 2E), which makes it likely that the members of the miR172 gene family are functionally interchangeable. Indeed, insertional mutations within either the miR172a-1 or miR172a-2 precursor gene do not cause any flowering-time or floral morphology defects (M.J. Aukerman, unpublished results), suggesting that there is genetic redundancy within the miR172 family. On the other hand, dicer-like1 mutants have lower overall levels of the entire miR172 family (Park et al., 2002; Kasschau et al., 2003) and display flowering-time and floral morphology defects that are opposite those of miR172 overexpression (Schauer et al., 2002). These observations provide independent evidence to support a role for miR172 in the regulation of flowering time and floral organ identity.
Although several different groups have identified plant miRNAs, the structure of any full-length plant miRNA precursor transcript was not known previously. We have found that EAT produces a 1.4-kb noncoding RNA that represents the full-length precursor of miR172a-2. The precursor transcript is polyadenylated and 5′ capped and appears to enter the splicing pathway, and these features strongly suggest that it is normally transcribed by RNA polymerase II (Pol II). It is unclear whether all or most of the other plant miRNA precursors also are Pol II transcripts. RNA polymerase III (Pol III) promoters have been used in mammalian systems to express hairpin precursors of miRNAs (McManus et al., 2002), and it is possible that some miRNA precursors are naturally transcribed by Pol III. Nevertheless, we have identified other plant miRNA sequences embedded within longer transcripts in EST collections (M.J. Aukerman, unpublished results), suggesting that many of the plant miRNA precursor transcripts are produced by Pol II rather than by Pol III.
RNA gel blot and immunoblot analyses (Figure 4A) indicated that miR172 downregulates its AP2-like target genes at the level of translation rather than by RNA cleavage. Although in animals there are several examples of translational regulation by miRNAs (Olsen and Ambros, 1999; Reinhart et al., 2000; Kawasaki and Taira, 2003), miR172 is the only plant miRNA that has been found to regulate its targets at the translational level. The identification of miR172-directed RNA cleavage products by PCR (Figure 4B) (Kasschau et al., 2003) appears to contradict this model of translational regulation; however, these cleavage products presumably represent a very small fraction of the total AP2 transcript population, because they are undetectable on RNA gel blots (Figure 4A). Furthermore, AP2 transcript abundance was unaffected by miR172a-2 overexpression, whereas AP2 was downregulated significantly (Figure 4A), indicating that regulation occurs at the level of translation. This unexpected result suggests that the regulation of target sequences by miRNAs may be more complex than was proposed previously, as we discuss further below.
It has been suggested that many plant miRNAs enter the RNA interference pathway exclusively as a result of their perfect or nearly perfect complementarity to putative targets (Rhoades et al., 2002; Tang et al., 2003). For example, miR171 has perfect complementarity to its SCARECROW-like target gene and has been shown to direct the cleavage of its target transcript (Llave et al., 2002b). A few mismatches between a miRNA and its target gene do not appear to abolish miRNA-directed RNA cleavage (Tang et al., 2003). In fact, most other plant miRNAs naturally have one, two, or three mismatches with their target genes; nonetheless, cleavage products from many of these target genes can be identified by PCR (Kasschau et al., 2003). Interestingly, miR172 itself contains only two mismatches with AP2 (Figure 2C), yet clearly it does not operate via an RNA cleavage mechanism. On the other hand, the fact that small amounts of RNA cleavage products do accumulate suggests overlap between the cleavage pathway and a second pathway operating at the translational level. Indeed, recent evidence suggests that miRNA-directed translational and RNA cleavage pathways may share some of the same molecular components (Hutvagner and Zamore, 2002; Mourelatos et al., 2002). It is possible that in the case of miR172 this overlap causes a small amount of target gene RNA cleavage as a side effect of translational regulation. In fact, it may turn out that other plant miRNAs normally regulate their targets at the translational level, even though cleavage products corresponding to their targets can be detected by PCR (Kasschau et al., 2003). Because these cleavage products are possible by-products of the proposed overlap between the two pathways and thus may not reflect a significant reduction of steady state target transcript levels, PCR-based detection of target RNA cleavage may give misleading results in these cases.
Although we have demonstrated that the regulation of miRNA target transcripts can occur at the level of translation in plants, the biochemical nature of the translational repression is not known. In C. elegans, lin-4 blocks the translation of its target transcript after the transcript has associated with ribosomes (Olsen and Ambros, 1999). Whether this postinitiation block also occurs in the case of miR172 and its AP2-like targets is unclear. Wheat germ extracts have been used to study miRNA-directed cleavage, and it may be possible to use such an in vitro system to characterize translational repression by miRNAs at the biochemical level. It also is unclear how miR172, which shares nearly perfect complementarity with its target, is directed away from the RNA cleavage pathway and into a translational repression pathway. We suggest a sequence determinant, within either miR172 or its target site, that functions as a recognition site for one or more of the components responsible for translational repression. This sequence determinant could be a single nucleotide, and in this regard it is interesting that miR172 is the only plant miRNA known to have an A residue at its 5′ end. As more plant miRNAs are discovered, it will be interesting to see if others begin with A and whether these also use a translational mode of regulation.
The target site for miR172 is found not only in AP2 but also in a few other AP2-like genes, including TOE1 and TOE2 (Figure 2D). Because ap2 mutants are not early flowering but miR172 overexpression lines are, we reasoned that miR172 controls flowering time by downregulating an AP2-like target other than AP2 itself. We have obtained data indicating that TOE1, and to a lesser extent TOE2, control flowering time and therefore are likely targets of miR172. Activation tagging of TOE1, followed by analysis of the toe1-2 loss-of-function mutant, indicated that TOE1 normally acts as a floral repressor (Table 1). These results are consistent with miR172 overexpression causing early flowering by downregulating TOE1. Interestingly, although toe2-1 was not significantly early flowering, the toe1-2 toe2-1 double loss-of-function mutant was earlier flowering than toe1-2, and this finding suggests that these two genes overlap in function. Further gene redundancy likely exists within this family of miR172 target genes, and we predict that triple and quadruple mutant combinations will more closely resemble the miR172 overexpression lines with regard to flowering time.
The loss-of-function phenotypes for TOE1 and TOE2 suggest that they are targets of miR172, and two additional pieces of evidence further solidify this connection. First, miR172 overexpression fully suppressed the late-flowering phenotype of TOE1 overexpression (Figure 5C), consistent with miR172 directly downregulating TOE1. Second, miR172-directed target site RNA cleavage products corresponding to TOE1 and TOE2 were recovered by PCR (Figure 4B) (Kasschau et al., 2003). Even though these cleavage products likely are side effects of translational control (see above), the fact that even a small amount of such cleavage products corresponding to these two target genes accumulate in wild-type plants strongly suggests that they normally are targeted by miR172. RNA cleavage products have been detected for all four predicted Arabidopsis miR172 targets (Kasschau et al., 2003), raising the possibility that any AP2-like gene containing the 21-nucleotide target site sequence, or a close variant, is a target of miR172. Indeed, the conservation of this sequence in a subset of AP2 genes from both monocots and dicots (Figure 2D) (M.J. Aukerman, unpublished results) strongly suggests that this sequence is a target site for miR172-like miRNAs in many plant species. This indicates that miR172 participates in flowering-time and floral organ identity control in plants other than Arabidopsis, which is a subject for future study.
Consistent with its proposed role in the regulation of flowering time, miR172 expression was regulated temporally (Figures 3D and 3E). Therefore, we propose a model (Figure 6) that predicts that the temporal upregulation of miR172 family members during seedling development leads to the temporal downregulation of TOE1 and TOE2 (and perhaps AP2 and/or TOE3). This model further predicts that flowering is triggered once the levels of these floral repressors decrease below a critical threshold. However, we cannot confirm that TOE1 and TOE2 are downregulated during this same period, because antibodies for these proteins are unavailable. Certain aspects of miR172, in particular its temporal expression pattern and its regulation of target genes at the translational level, are reminiscent of lin-4 and let-7, two miRNAs that control developmental timing in C. elegans (Feinbaum and Ambros, 1999; Reinhart et al., 2000). The fact that miR172 also affects timing in plants (Figure 1B, Table 1) indicates that both plants and animals use miRNAs to control temporal events during development.
Model Depicting the Temporal Regulation of Flowering by miR172.
In this model, the temporal expression of miR172 causes temporal downregulation of the AP2-like target genes at the level of translation, which triggers flowering once the target proteins decrease below a critical threshold (dotted line). The onset of floral initiation is indicated by the black bar.
Flowering time in Arabidopsis is controlled by endogenous and environmental mechanisms, and there are many genes implicated in the regulation of flowering time (reviewed by Simpson and Dean, 2002). These genes have been assigned to several pathways based on their response to photoperiod, vernalization, and hormones. Although it is clear that at least some of the regulation of these pathways is performed by transcription factors, most notably those of the MADS box family, no evidence has been presented to date of any involvement of AP2 family members in this process or of miRNAs. Here, we provide evidence for the involvement of both miRNAs and AP2-like proteins in flowering time. Although we have not yet assigned miR172 to a specific flowering-time pathway, we found that lines overexpressing miR172 have a normal response to photoperiod (Table 1). Furthermore, mutations that affect either the autonomous (luminidependens) or photoperiod (constans) pathways did not change the levels of miR172 (Figure 3C), suggesting that it is not a downstream component of these pathways. It is possible that miR172 and its targets function within a flowering-time pathway that is poorly characterized, such as the pathway that regulates flowering in response to intrinsic developmental age (Simpson and Dean, 2002) or the ambient-temperature-response pathway (Blazquez et al., 2003). Future physiological experiments with miR172 overexpression lines and the AP2-like loss-of-function mutants, coupled with transcript analysis of other flowering-time genes, should allow a more precise placement of miR172 within the flowering pathway network and help to identify downstream components. Interestingly, certain flowering-time genes, such as FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CO1, display a temporal expression pattern similar to that of miR172 and thus are potentially downstream of miR172 and the AP2-like floral repressors.
Plant Material and Growth Conditions
All experiments were performed on the Columbia (Col-0) ecotype of Arabidopsis thaliana. Plants were grown in long days (16 h of light, 8 h of dark) under cool-white light at 22°C, except where noted.
Activation Tagging
Arabidopsis plants were transformed by a modified version of the floral-dip method (Clough and Bent, 1998) in which Agrobacterium tumefaciens cell suspension was applied to plants by direct watering from above. The T-DNA vector used, pHSbarENDs, contained four copies of the 35S enhancer of Cauliflower mosaic virus adjacent to the right border, an arrangement similar to that described by Weigel et al. (2000). Transformed plants were selected with glufosinate (Basta) and screened for flowering time, which resulted in the identification of the early-flowering mutant eat-D. A single T-DNA cosegregating with early flowering was identified in eat-D, and thermal asymmetric interlaced PCR was performed to amplify sequences adjacent to the left and right borders of the T-DNA. To identify transcripts upregulated in eat-D, we probed RNA gel blots containing RNA extracted from wild-type (Col-0) and eat-D plants. Probes for the genes At5g04270 and At5g04280 (Figure 2A) detected no difference between the wild type and eat-D, whereas a probe from the intergenic region identified an ∼1.4-kb transcript that was expressed at significantly higher levels in eat-D than in the wild type. 5′ and 3′ rapid amplification of cDNA ends PCR was performed to isolate the full-length EAT cDNA using the GeneRacer kit (Invitrogen, Carlsbad, CA) and RNA extracted from wild-type floral buds. The fact that the GeneRacer protocol allows selective amplification of 5′ capped messages as well as the use of oligo(dT) to prime first-strand cDNA synthesis indicated that the EAT transcript is normally 5′ capped and polyadenylated.
We amplified the entire 1.4-kb EAT transcription unit from Col-0 genomic DNA using primers located at the 5′ and 3′ ends of the gene that also contained XhoI sites (5′-GACTACTCGAGCACCTCTCACTCCCTTTCTCTAAC-3′ and 5′-GACTACTCGAGGTTCTCAAGTTGAGCACTTGAAAAC-3′). To generate the 35S-EAT construct, the XhoI-cut EAT was inserted into the XhoI site of binary vector pBE851 between the 35S promoter of Cauliflower mosaic virus and a β-phaseolin terminator. The 35S-eatdel construct was generated as follows. The 1.4-kb EAT cDNA (in a modified pBluescript SK+ vector [Stratagene] lacking BamHI and HindIII sites) was digested with BamHI and HindIII, releasing a 159-bp fragment containing the miR172a-2 sequence and hairpin precursor. The vector fragment containing the EAT cDNA with the microRNA (miRNA) and hairpin sequences deleted was gel-purified. Oligonucleotides that reconstructed the entire 159-bp fragment except for the 21-nucleotide miRNA sequence were annealed and ligated into the digested EAT cDNA, generating a modified EAT cDNA lacking the 21-nucleotide miRNA sequence. This eatdel cDNA was ligated into pBE851 as described above.
To generate 35S-miR172a-1, primers were designed to amplify a 1.9-kb genomic fragment surrounding miR172a-1 (5′-CCGCTCGAGATCTGTGTTCTAGATCTCACCAGGTC-3′ and 5′-CCGCTCGAGGAGTGAAGACATATTATATTAATTGAGGTAC-3′). This fragment encompasses a full-length cDNA sequence, RAFL21-01-E23, deposited in GenBank. The amplified fragment was digested with XhoI and inserted into pBE851 as described above. To generate the 35S-TOE1 construct, a 2-kb genomic fragment containing the entire TOE1 coding region was amplified with primers containing KpnI sites at the ends (5′-CCGGTACCATGTTGGATCTTAACCTCAACGCTGATTC-3′ and 5′-CCGGTACCTTAAGGGTGTGGATAAAAGTAACCACGTGTTG-3′). After KpnI digestion of the PCR product, the KpnI sites were converted to XhoI sites by ligation of an eight-nucleotide adapter oligonucleotide, and the fragment was ligated into XhoI-cut pBE851. All constructs were transformed into Agrobacterium, and Col-0 was transformed with the Agrobacterium strains by floral dip (Clough and Bent, 1998).
Analysis of miR172 and AP2 Expression
Total RNA was isolated from wild-type and eat-D plants at the stages and tissues indicated in the figure legends using TRIZOL reagent (Sigma). Fifty micrograms of each RNA was subjected to electrophoresis on a 15% TBE-Urea Criterion gel (Bio-Rad) and electroblotted onto Hybond-N+ filter paper (Amersham) using a TransBlot-SD apparatus (Bio-Rad). The filter then was hybridized at 37°C overnight in UltraHyb-Oligo buffer (Ambion, Austin, TX) with 32P-labeled oligonucleotides. The oligonucleotides were 30-mers that corresponded to either the sense or antisense strand of miR172a-2, with four or five nucleotides of flanking sequence on each side (5′-ATATGAGAATCTTGATGATGCTGCATCAAC-3′ and 5′-GTTGATGCAGCATCATCAAGATTCTCATAT-3′). The filters were washed twice at 37°C in buffer containing 2× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate) and 0.5% SDS. For S1 analysis, probe was made by end-labeling an oligonucleotide (5′-ATGCAGCATCATCAAGATTCTCATATACAT-3′) with T4 polynucleotide kinase and 32P. Hybridization and processing of S1 reactions were performed using standard protocols (Ausubel et al., 1995).
RNA gel blot analysis to determine AP2 levels was performed on poly(A)-enriched RNA from floral buds. The RNA was subjected to electrophoresis on 5% TBE-Urea Criterion gels, blotted as described above, and incubated with 32P-labeled probes specific for AP2, TOE1, TOE2, EAT, or ACT11. The probes were amplified by PCR using Col-0 genomic DNA as template with the following primers: AP2 (5′-TTTCCGGGCAGCAGCAACATTGGTAG-3′ and 5′-GTTCGCCTAAGTTAACAAGAGGATTTAGG-3′); TOE1 (5′-TTTCCGGCCACAACCTCCCAATGACAATG-3′ and 5′-TTTTGAAAGGGGACTAGAGTGTTGAGAG-3′); TOE2 (5′-TACACGCCCTCCTTCATCCACAGCTATTC-3′ and 5′-GAAACTACAGCTTGTGTGCTGAGCCAAAC-3′); and ACT11 (5′-ATGGCAGATGGTGAAGACATTCAG-3′ and 5′-GAAGCACTTCCTGTGGACTATTGATG-3′). The EAT probe fragment was isolated by digesting the EAT cDNA in pBluescript SK+ (see above) with XhoI and gel-purifying the insert. Basic Local Alignment Search Tool (BLAST) searches with the sequences contained in each AP2-like gene probe indicated that each probe was gene specific. UltraHyb buffer was used for hybridization to the AP2-like genes (Ambion), and subsequent processing was as described in the UltraHyb protocol. Immunoblot analysis of AP2 was performed on proteins extracted from floral buds. After electrophoresis on a 10% SDS-PAGE gel, proteins were transferred to a Hybond-P membrane (Amersham) and incubated with an antibody specific for AP2 (aA-20; Santa Cruz Biotechnology, Santa Cruz, CA). The AP2 antibody specifically recognizes an ∼45-kD protein that corresponds to AP2, because in an ap2 null mutant this 45-kD protein is absent (Chen, 2003). The blot was processed using the ECL-Plus kit (Amersham).
Identification of toe1-1D, toe1-2, and toe2-1
toe1-1D was identified in the same manner as described for eat-D above, except that semiquantitative reverse transcriptase–mediated (RT) PCR was used to confirm the overexpression of TOE1 in toe1-1D (Figure 3B). Oligonucleotides used to detect TOE1 expression were 5′-CAAATGTGGTAGATGGGAAGCTAGGATG-3′ and 5′-CCAGTTACTCATCATCCCTTCAGCTTCAC-3′. RT-PCR of TOE1 was performed for 22 cycles to ensure logarithmic amplification. Control primers for ACT11 were 5′-ATGGCAGATGGTGAAGACATTCAG-3′ and 5′-GAAGCACTTCCTGTGGACTATTGATG-3′. RT-PCR products were subjected to electrophoresis on 2% agarose gels, blotted onto Hybond-N+, and hybridized with 32P-labeled probes for TOE1 and ACT11. toe1-2 (SALK_069677) and toe2-1 (SALK_065370) were identified in the collection of Salk mutants available from the Arabidopsis Stock Center. PCR amplification was used to confirm the presence of T-DNA inserts within both genes.
Upon request, materials integral to the findings presented in this publication will be made available in a timely manner to all investigators on similar terms for noncommercial research purposes. To obtain materials, please contact M.J. Aukerman, milo.j.aukerman{at}usa.dupont.com.
The GenBank accession number for RAFL21-01-E23 is AK118705.
NOTE ADDED IN PROOF
Recently, a microRNA controlling leaf morphogenesis was described (Palatnik, J.F., Allen, E., Wu, X., Schommer, C., Schwab, R., Carrington, J.C., and Weigel, D. [2003]. Control of leaf morphogenesis by microRNAs. Nature 425, 257–263).
We thank M. Mucha for technical assistance; W. Park, S. Tingey, S. Martino-Catt, and M. Albertsen for helpful discussions; and K. Glassman for critical reading of the manuscript. We also thank the anonymous reviewers for their comments.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.016238.
Accepted September 10, 2003.
Published October 10, 2003.
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (1995). Current Protocols in Molecular Biology. (New York: John Wiley & Sons).
Blazquez, M.A., Ahn, J.H., and Weigel, D. (2003). A thermosensory pathway controlling flowering time in Arabidopsis thaliana. Nat. Genet. 33, 168–171.
Bowman, J.L., Smyth, D.R., and Meyerowitz, E.M. (1991). Genetic interactions among floral homeotic genes of Arabidopsis. Development 112, 1–20.
Brennecke, J., Hipfner, D.R., Stark, A., Russell, R.B., and Cohen, S.M. (2003). bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell 113, 25–36.
Chen, X. (2003). A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science, in press.
Chuck, G., Meeley, R.B., and Hake, S. (1998). The control of maize spikelet meristem fate by the APETALA2-like gene indeterminate spikelet1. Genes Dev. 12, 1145–1154.
Clough, S.J., and Bent, A.F. (1998). Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743.
Feinbaum, R., and Ambros, V. (1999). The timing of lin-4 RNA accumulation controls the timing of postembryonic developmental events in Caenorhabditis elegans. Dev. Biol. 210, 87–95.
Grishok, A., Pasquinelli, A.E., Conte, D., Li, N., Parrish, S., Ha, I., Baillie, D.L., Fire, A., Ruvkun, G., and Mello, C.C. (2001). Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106, 23–34.
Hutvagner, G., McLachlan, J., Pasquinelli, A.E., Balint, E., Tuschl, T., and Zamore, P.D. (2001). A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 293, 834–838.
Hutvagner, G., and Zamore, P.D. (2002). A microRNA in a multiple-turnover RNAi enzyme complex. Science 297, 2056–2060.
Kasschau, K.D., Xie, Z., Allen, E., Llave, C., Chapman, E.J., Krizan, K.A., and Carrington, J.C. (2003). P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA function. Dev. Cell 4, 205–217.
Kawasaki, H., and Taira, K. (2003). Hes1 is a target of microRNA-23 during retinoic-acid-induced differentiation of NT2 cells. Nature 423, 838–842.
Ketting, R.F., Fischer, S.E., Bernstein, E., Sijen, T., Hannon, G.J., and Plasterk, R.H. (2001). Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev. 15, 2654–2659.
Lagos-Quintana, M., Rauhut, R., Lendeckel, W., and Tuschl, T. (2001). Identification of novel genes coding for small expressed RNAs. Science 294, 853–858.
Lagos-Quintana, M., Rauhut, R., Yalcin, A., Meyer, J., Lendeckel, W., and Tuschl, T. (2002). Identification of tissue-specific microRNAs from mouse. Curr. Biol. 12, 735–739.
Lau, N.C., Lim, L.P., Weinstein, E.G., and Bartel, D.P. (2001). An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294, 858–862.
Lee, R.C., and Ambros, V. (2001). An extensive class of small RNAs in Caenorhabditis elegans. Science 294, 862–864.
Lee, R.C., Feinbaum, R.L., and Ambros, V. (1993). The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843–854.
Lee, Y., Jeon, K., Lee, J.T., Kim, S., and Kim, V.N. (2002). MicroRNA maturation: Stepwise processing and subcellular localization. EMBO J. 21, 4663–4670.
Llave, C., Kasschau, K.D., Rector, M.A., and Carrington, J.C. (2002a). Endogenous and silencing-associated small RNAs in plants. Plant Cell 14, 1605–1619.
Llave, C., Xie, Z., Kasschau, K.D., and Carrington, J.C. (2002b). Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 297, 2053–2056.
McConnell, J.R., Emery, J., Eshed, Y., Bao, N., Bowman, J., and Barton, M.K. (2001). Role of PHABULOSA and PHAVOLUTA in determining radial patterning in shoots. Nature 411, 709–713.
McManus, M.T., Petersen, C.P., Haines, B.B., Chen, J., and Sharp, P.A. (2002). Gene silencing using micro-RNA designed hairpins. RNA 6, 842–850.
Moose, S.P., and Sisco, P.H. (1996). Glossy15, an APETALA2-like gene from maize that regulates leaf epidermal cell identity. Genes Dev. 10, 3018–3027.
Mourelatos, Z., Dostie, J., Paushkin, S., Sharma, A., Charroux, B., Abel, L., Rappsilber, J., Mann, M., and Dreyfuss, G. (2002). miRNPs: A novel class of ribonucleoproteins containing numerous microRNAs. Genes Dev. 16, 720–728.
Okamuro, J.K., Caster, B., Villarroel, R., Van Montagu, M., and Jofuku, K.D. (1997). The AP2 domain of APETALA2 defines a large new family of DNA binding proteins in Arabidopsis. Proc. Natl. Acad. Sci. USA 94, 7076–7081.
Olsen, P.H., and Ambros, V. (1999). The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation. Dev. Biol. 216, 671–680.
Park, W., Li, J., Song, R., Messing, J., and Chen, X. (2002). CARPEL FACTORY, a Dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Curr. Biol. 12, 1484–1495.
Pickford, A.S., and Cogoni, C. (2003). RNA-mediated gene silencing. Cell. Mol. Life Sci. 60, 871–882.
Reinhart, B.J., Slack, F.J., Basson, M., Pasquinelli, A.E., Bettinger, J.C., Rougvie, A.E., Horvitz, H.R., and Ruvkun, G. (2000). The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403, 901–906.
Reinhart, B.J., Weinstein, E.G., Rhoades, M.W., Bartel, B., and Bartel, D.P. (2002). MicroRNAs in plants. Genes Dev. 16, 1616–1626.
Rhoades, M.W., Reinhart, B.J., Lim, L.P., Burge, C.B., Bartel, B., and Bartel, D.P. (2002). Prediction of plant microRNA targets. Cell 110, 513–520.
Schauer, S.E., Jacobsen, S.E., Meinke, D.W., and Ray, A. (2002). DICER-LIKE1: Blind men and elephants in Arabidopsis development. Trends Plant Sci. 11, 487–491.
Simpson, G.G., and Dean, C. (2002). Arabidopsis, the Rosetta stone of flowering time? Science 296, 285–289.
Slack, F.J., Basson, M., Liu, Z., Ambros, V., Horvitz, H.R., and Ruvkun, G. (2000). The lin-41 RBCC gene acts in the C. elegans heterochronic pathway between the let-7 regulatory RNA and the LIN-29 transcription factor. Mol. Cell 5, 659–669.
Tang, G., Reinhart, B.J., Bartel, D.P., and Zamore, P.D. (2003). A biochemical framework for RNA silencing in plants. Genes Dev. 17, 49–63.
Weigel, D., et al. (2000). Activation tagging in Arabidopsis. Plant Physiol. 122, 1003–1013.
Wightman, B., Ha, I., and Ruvkun, G. (1993). Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 75, 855–862.
Xu, P., Vernooy, S.Y., Guo, M., and Hay, B.A. (2003). The Drosophila microRNA Mir-14 suppresses cell death and is required for normal fat metabolism. Curr. Biol. 13, 790–795.
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The Plant Cell Nov 2003, 15 (11) 2730-2741; DOI: 10.1105/tpc.016238
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Intel tried to push back at coverage of a recently published paper that found its Core i7 processors couldn't match the parallel processing performance of an Nvidia GPU, saying its rival took the findings of the paper out of context in a blog post that trumpeted the results.
"Nvidia picked up on a small part of the paper and took it out of context," said Nick Knupffer, an Intel spokesman, in an e-mail.
However, the paper's findings were clear. Titled "Debunking the 100x GPU vs. CPU myth: An evaluation of throughput computing on CPU and GPU," the paper was written by Intel engineers and sought to discredit claims that GPUs outperform CPUs by a wide margin in parallel processing applications. Nevertheless, the paper still found that one of Intel's fastest quad-core desktop processors, the 3.2GHz Core i7 960, was markedly slower than an older Nvidia graphics card, the GeForce GTX280, in benchmark tests conducted by the engineers.
"Our data shows that GTX280 only has an average of 2.5X performance advantage over Core i7 in the 14 kernels tested," the paper said, noting that the Nvidia GPU was more than 14 times faster under some circumstances.
Nvidia discussed the paper's findings in a blog post that questioned the magnitude of the GPU performance advantage described in Intel's paper, providing links to 10 customers who documented performance increases of 100 times or more using software that was optimized to run on GPUs.
The company also noted Intel researchers didn't publish the software code and data they used to conduct the benchmark tests. Without that information, it's impossible to repeat Intel's tests to confirm the paper's findings or to understand how the code was optimized for the chips.
"It's just bad science," said Andy Keane, general manager of GPU computing at Nvidia and the author of the blog post, discussing the paper in an interview.
Despite their rivalry, Intel and Nvidia readily agree on the importance of parallel processing capabilities. The only disagreement between them is whether the CPU or GPU is the component that's best suited to handling these parallel processing workloads, an argument largely based on their respective product strengths.
Intel, which dominates the market for x86 CPUs, believes that its chips have the necessary power to handle these workloads, whereas Nvidia -- the world's largest provider of discrete, or standalone, graphics chips -- thinks using the GPU is a better choice. Advanced Micro Devices, which sells both GPUs and x86 CPUs, shares Nvidia's view that GPUs can offer a significant performance boost in certain types of applications.
The Intel paper generally reinforces the position held by Nvidia and AMD by acknowledging that the GTX280 holds a performance advantage over the Core i7 960 when it comes to parallel processing.
The reason for the difference in performance between CPUs and GPUs is that the two chips are designed with fundamentally different purposes in mind. CPUs are designed to handle a series of instructions in sequential order. The addition of multithreading capabilities and multiple cores gives CPUs a significant degree of parallelism, but that pales in comparison to what GPUs from Nvidia and AMD are capable of with certain applications, such as video encoding.
Originally used to draw thousands of polygons and map textures for realistic computer graphics, GPUs are parallel processors designed to handle many operations at the same time. Each GPU processor core may be simpler and less powerful than a CPU core but the latest Nvidia GPUs have hundreds of them, compared to just four cores on the Core i7 960. The ability to process hundreds of operations simultaneously is where GPUs get their parallel processing advantage -- a point that Intel researchers acknowledged in their paper.
"There is little doubt that today's CPUs would provide the best single thread performance for throughput computing workloads. However, the limited number of cores in today's CPUs limits how many pieces of data can be processed simultaneously. On the other hand, GPUs provide many parallel processing units which are ideal for throughput computing," the paper said.
However, hardware alone isn't enough. Getting the most performance requires that an application be optimized to run on a GPU. That can be time-consuming and expensive. As a result, most applications optimized to run on GPUs are typically used in financial analysis and other fields, such as oil exploration and scientific research, where the benefits from higher performance outweigh the added cost of optimization.
GPU acceleration capabilities are increasingly a feature of consumer and business software.
One of the first companies to use the technology was Apple, which added the ability to use GPUs to handle some application workloads in the latest release of MacOS X, called Snow Leopard. Other companies followed suit, including Adobe, which uses GPU acceleration in Premiere Pro CS5, After Affects CS5 and Photoshop CS5.
Microsoft included GPU acceleration capabilities in Windows 7 and is adding the technology to its upcoming Internet Explorer 9 browser.
Smaller software companies are also attracted to GPU acceleration, like Fotegrafik Labs, a Singaporean startup that developed software used to search a database of images or video files.
The image search software was developed on a server that's powered by a single quad-core Intel Xeon processor, but Fotegrafik Labs is considering switching to GPUs because they are more efficient than CPUs for this type of application and would allow the software to be used with very large databases, said Ori Cohen, the company's CEO, citing the relatively low cost of GPUs compared to the high performance they offer.
"If Intel could provide us with a CPU with hundreds of cores at a competitive price, then maybe the CPU would be an alternative to the GPU for video applications," Cohen said.


A first time buyer from Telford has recently moved into a new home at Lovell's The Oaklands development in Dawley.
25 year old Alex McClony has recently bought a three bedroom semi-detached Westwood house style at The Oaklands development as his first home.
After renting for many years, Database Administrator Alex decided to take the next step and purchase his own home in Telford, using the Government backed Help to Buy scheme.
Alex said: "I am from the area and I wanted somewhere that is my own. This was the perfect size, and with the Help to Buy: Equity Loan scheme it was very affordable which was the main selling point for me.
"I loved the house from the start, especially the ensuite, garden and garage. I have found that being new build the property is much more energy efficient than my previous home, which is also a bonus.
Alex wasn't initially searching for a new build, however the affordability of homes and location of The Oaklands development was too hard to deny. Located on Matlock Avenue, The Oaklands is a delightful development of 44 two and three bedroom homes.
Alex lives in a Westwood style home, which includes a double oven, integrated fridge freezer, vinyl flooring in the kitchen and bathroom, a built in wardrobe in bedroom one, and more added qualities.
Perfect for first time buyers, Lovell offers the best specification of houses in the Telford area. With excellent transport links and surroundings, The Oaklands is the ideal place for commuters and young professionals, as well as young families looking to settle.
With prices starting from £129,950, the homes are available to purchase with the Help to Buy: Equity Loan scheme. The scheme allows homebuyers to buy a new home under the value of £600,000 with just a 5% deposit and 75% mortgage, with the final 20% made up by an equity loan provided by the Government. First time buyers looking for their new home in Telford only need a deposit of £6,498 for a house priced at £129,950.
To book your viewing of the show homes, please call 01952 700 655 or email the sales team on sales.midlands@lovell.co.uk.


* A book to transform the performance of all clay shooters * Proven tournament techniques * Written by one of the world's leading instructors Breaking Clays is a comprehensive and practical book that presents in- depth advice and instruction for shooters of all disciplines. Beginning with the basics and advancing to proven tournament techniques, the book is packed with invaluable tips on how to break more clays in your chosen game. Chris Batha has worked with some of the top competitors and shooting coaches in the world today., While every top shot has his own approach to shooting and teaching, Chris recognizes that what works for one person will not necessarily work for another. This clear and concise book offers a distillation of the best tips and techniques that really work to improve your scores and give you the knowledge to develop to your full shooting potential.
Want to know when Breaking Clays : Target Tactics, Tips and Techniques - nzgameshop.com drops below a certain price? Enter your email address and price below and we'll let you know when it drops below that price!


text:
HTC Android 5.0 Lollipop rollout is now officially late
By Jon Mundy
Contact via linkedin February 2, 2015 8:28 am GMT
The Android 5.0 Lollipop update is now officially late for many HTC One (M8) and HTC One (M7) owners, according to the company.
The Taiwanese manufacturer previously set itself the goal of providing updates to the latest version of the Android OS within 90 days of Google making the final code available. However, in the case of Android 5.0 Lollipop, HTC will not be able to stick to that target.
At least, it won't in many cases. The company has provided an update on its progress, and has been open about its struggles.
HTC has managed to provide Lollipop updates to Google Play, Unlocked, and Developer Editions of the HTC One (M8), but that leaves many that will no receive the update on time.
"Despite everyone's best efforts some carrier versions of the HTC One (M8) and HTC One (M7) will not meet our 90 day goal, which is February 1st," says HTC vice president of product management Mo Versi.
Apparently, it's all Google's fault. "If you've been following the progress of this rollout you will know that Google has had to address several issues with this release," says Versi.
Read More: Android 5.0 Lollipop update: When will my phone get it
HTC has apparently been "diligently working" with Google to fix some of these issues at Google's end, as well as to implement Google's own fixes. The end result, however, is that the company's self-imposed 90-day deadline has passed with many HTC One (M8) and (M7) owners not getting their Lollipop update.
As the post points out, HTC has led the way with the speed and transparency of its update process in the past, and by all accounts Google has indeed released a somewhat unfinished, buggy release in Android 5.0. Just ask Motorola.
Contact via linkedin
Jon is a seasoned freelance writer who started covering games and apps in 2007 before expanding into smartphones and consumer tech, dabbling in lifestyle and media coverage along the way. Besides bein…

redpajama_set_name: RedPajamaCommonCrawl


text:
Posted by Deborah Gimblett
Frequently Asked Questions about the project, UNICEF and Rotary
Q: In recent years, there has been a growing anti-vaccine sentiment in some communities where parents refuse to vaccinate their children. What is UNICEF's reaction?
A: Immunisation saves lives and gives children the opportunity to live a healthy life and to reach their full potential. Children who are not immunised face illness, disability and death.
Immunisation is extremely safe, with very low risk of serious complications or allergic reactions. Erroneous claims about vaccination (that it causes autism, contains harmful "toxins" or damages fertility) are entirely unscientific and false.
When immunisation rates against highly contagious diseases like measles fall below 95%, "herd immunity" breaks down, creating a higher chance for intense transmission and disease outbreaks – with dire consequences for children. Herd immunity also protects babies who are too young to be vaccinated (less than six months) against measles and anyone who is immunosuppressed.
All children, no matter where they live or their circumstances, have the right to survive and to access the best available healthcare – and we each have the responsibility to help them realize these rights. In the countries where UNICEF operates, we work with communities to raise awareness about the importance of vaccines.
Q: Is UNICEF transparent? How do we know the funds raised will be spent in the way promised?
A: UNICEF is a signatory to the international transparency trade initiative.
In 2016, the Aid Transparency Index placed UNICEF in the 'very good' category, rated 3/46 organisations. In 2015 UNICEF launched a transparency microsite showing exactly where and when funds raised are spent, which can be found here: http://open.unicef.org/
UNICEF Australia and New Zealand will produce annual reports showing where the funds raised have been spent and the difference this is making. There will also be opportunities for Rotary Representatives to go to the Pacific to see the project first hand to enable them to report back to fellow Rotarians.
Q: Is the Rotary: Give Every Child A Future Program sustainable?
A: One of UNICEF's key priorities in programme design is sustainability. UNICEF works in partnership with governments to design 5-year country plans, and our programmes fit within this plan. As in this case, UNICEF will set up this program in a way that allows the governments to take over its implementation at the end of the initial funding. Therefore, the impact of setting up the relevant infrastructure, training healthcare workers and working with local communities to encourage them to have their children immunised will have long lasting benefits. The savings to the national healthcare budgets that will arise through lower hospitalisation rates and lower incidence of these diseases will contribute to the ongoing costs of the immunisation program.
Q: Will this project eliminate these diseases totally, like the Polio Plus project aims to do for polio?
A: The short answer is no, or at least certainly not in the short to medium term. These viruses are too prevalent and mutate too easily. They are more akin to the influenza virus. It would be very difficult, if not impossible, to eliminate them totally. So, the aim is to provide everyone with a degree of protection so that if they do get infected then the body's immune system can easily deal with the virus.
Q: Are there any side-effects from these vaccines? If so, what are they and how serious can they be?
A: The answer varies for the three vaccines.
For the rotavirus vaccine recipients may have slightly increased risk of developing mild diarrhoea or vomiting in the week after the vaccine administration. Serious side effects are very rare. There is also a slightly increased risk of intussusception, a rare form of bowel blockage, associated with the rotavirus vaccine. However, the risks of rotavirus gastroenteritis are many times greater than the very small risk of immunisation. Before rotavirus vaccine became available in Australia, almost every child was infected by rotavirus by the age of 5 years. About 10,000 young children were hospitalised with rotavirus gastroenteritis each year and up to one young child a year died from complications. Since rotavirus vaccination started in 2007 more than 7,000 hospital admissions for rotavirus are prevented each year. Those who do go to see a doctor are less ill. This compares with more than 200 babies being hospitalised for intussusception each year, of which only about an extra 14 may be related to having received rotavirus vaccine. Based on the established benefits of rotavirus vaccination and the rare occurrence of IS, both the World Health Organization (WHO), the Australian Technical Advisory Group on Immunisation (ATAGI) have recommended the continued use of rotavirus vaccine for infants under the National Immunisation Program (NIP).
The pneumococcal vaccine is very safe, although, like all vaccinations, it has some side effects. It is not possible to catch a pneumococcal infection from the vaccine, as the vaccine doesn't contain any live bacteria. Mild side effects of the pneumococcal conjugate vaccine (PCV), which is the version of the pneumococcal vaccine given to babies under the age of two, include:
a slightly raised temperature
redness at the site of the injection
hardness or swelling at the site of the injection
feeling sleepy
not sleeping well
More serious side effects of the PCV vaccine are rare, and include:
high temperature, possibly leading to convulsions (febrile seizures)
allergic itchy skin rash
With the HPV vaccine, some people might experience mild adverse effects typical of other injected vaccines: soreness, swelling, redness at the injection site, mild temperature or feeling faint. The World Health Organization's Global Advisory Committee on Vaccine Safety continues to review the accumulating data on HPV vaccine safety and finds that the vaccine is safe, with over 10 years of data and over 200 million doses given worldwide.
Q: HPV is spread primarily through sexual activity. How do we overcome the taboos in some societies to discussing such matters, particularly when we are planning to vaccinate the girls in their early teenage years? Will vaccinating the girls somehow be seen as giving them permission to become sexually active?
A: The first point that can be made is that the HPV vaccine is not a contraceptive. What it is doing is providing them with protection again various cancers, particularly the most common forms of cervical cancer. It is true that they are unlikely to become infected unless and until they become sexually active. However, it is also true that the protection is much more effective if given before the person becomes infected. So, the target group is the 12-15 year old girls but this should not be seen as in any way encouraging them to become sexually active, indeed quite the opposite. Of course, this conversation has to be handled appropriately in each country in the context of the social attitudes of that society. To help address this issue, the program includes social mobilisation activities before the introduction of HPV vaccine in each country. These activities help communities, local leaders and religious groups to gain a healthy understanding of the HPV vaccine and what immunisation means.
Q: In Australia and New Zealand, the boys are also given the HPV vaccine. Why are boys not included in this program?
A: It is essentially a question of available resources and setting priorities. The recommendation from WHO and others is that we should give priority to the girls. Vaccinating the boys will give them protection against some relatively rare cancers and, perhaps more importantly, it can also restrict transmission of the virus. However, the HPV vaccine is the most expensive of the three vaccines that we will be distributing and therefore it would add substantially to the cost of the project to extend the program to include the boys. There is also a question of sustainability as not all counties may be able to afford the continuing cost if the boys were included. Of course, it would be great to be able to vaccinate the boys as well, but the girls are the priority.
Q: As a Rotarian how can I help and will there be an opportunity to visit some of the islands?
A: Firstly, we need to be clear that this is not a project where we need to mobilise a large group of people from Australia, New Zealand or elsewhere to go into the Pacific to vaccinate the children. For this program to be sustainable it is critical that the implementation be left in the hands of the local people. This project is all about increasing the capabilities of these countries to look after themselves. However, there will be opportunities for some Rotarians to visit and see first-hand what is happening. There will likely be a need to provide people who can assist in the training of healthcare and community workers – a train-the-trainer type program. The people required for this may need to have particular skills and qualifications. For the majority of Rotarians, the best way that they can help is by playing a part in raising the necessary money to fund the project, raising awareness of the issues and by using the project to promote Rotary and the work that we do.
Q: Why does Rotary need to raise money for this, surely it is the responsibility of government?
A: Rotarians see a problem and work to find a solution. Yes, governments and others could, and maybe should, take the responsibility. In the case of many Pacific Island governments they simply do not have the resources. Like so many things in the past Rotary needs to be the catalyst.
Q: In your promotion material you say $45 will contribute to immunise 1 child, $450 for 10 children, $4,500 for 100 etc. How do you arrive at these figures?
A: These figures are approximations and an average to let individuals, clubs, districts and other organisations know the scale to which they can help. Rotary is trying to raise $4.5 – 5 million Australian/New Zealand dollars and this will benefit 100,000 children over the 3-4 year life of this project – thus approximately $45 per child. Every dollar collected goes to the programme. Whether you think in New Zealand Dollars or Australian Dollars, please help.
To see a slide presentation on Give Every Child a Future, click here.
If you have any other questions please direct to: everychildafuture@gmail.com


Contacts are essential elements in any device. Everyone needs contacts on his phone or computer in other to be connected with his friends or family. If you are using an iPhone, you can sync your contacts easily with iCloud. However, some settings need to be enabled on your phone to do this. On the other hand, if you have contacts in your Google Drive or Gmail, you can also sync them. Syncing contacts from Google drive is a simple task as well. So many times it happens when you transfer your contacts from one device to another or from one account to another. You cannot copy paste all the contacts one by one. So you will need to know some methods if you want to import Gmail contacts to iCloud account.
Today we will tell you how to transfer Google contacts to your iCloud account. Both of these accounts are entirely different. So, most of the people think that they cannot do this job. However, you will need a computer or Mac to do this job. In order to import Google contacts to iCloud account, you will need to first save the contacts in your system and then import your Gmail contacts to your iCloud account. The whole process is straightforward and just takes a few minutes. Here is how you can do this.
Before importing Gmail contacts, you will need to have a copy of your contacts on your computer. The only way to do this is logging in your Google account. So follow the process below to import contacts on your computer.
Step 2: Now sign in your Google account by providing email and password.
Step 3: On your Google accounts screen, locate the option named More on the top.
Step 4: Now tap on export. You will see a couple of options here including the types of contacts that needs to be imported.
Step 5: Select the vcf file format from the list.
Step 6: Now you can save the files on your computer.
The second procedure is importing the contacts in iCloud which are on your computer. The process is quite similar to the previous one. You will need to open your iCloud account and then import Gmail contacts to iCloud account. Have a look at the second procedure below.
Step 1: Go to iCloud.com and login to your account.
Step 2: Now open the contacts in your iCloud account.
Step 3: In the bottom left corner, click on More option. You will see a dropdown menu with different options.
Step 4: Click on Import vCard option from the list.
Step 5: Select the contacts that you imported in your computer from the Google contacts.
You are all done! The contacts from your Google account has been imported to your iCloud account successfully. Now you can use the contacts from your iCloud account as well.


A chemistry book that teaches you to think using design and narrative.
568 backers pledged $11,321 to help bring this project to life.
You'll be emailed a high-quality PDF of the full book.
You'll be emailed the PDF of the book + 2) as well as a Kickstarter-exclusive handbook about perspectives on graphic design and teaching, motivations behind creating the project, and the principles used to write and design the book.
You'll be emailed the PDF and handbook + 3) and have your name listed in the opening acknowledgements section of the physical book.
I just added this reward because of messages I've received. You will be emailed the PDF and the handbook, and you will receive the physical book. I will thank you on the website, because unfortunately there is no more room in the book!
You'll be emailed the PDF and the handbook, thanked in the book + 4) you will receive the physical book, along with 5) a Kickstarter-exclusive pKa table bookmark.
You'll be emailed the PDF and the handbook, thanked in the book + 4) you will receive the physical book, along with 5) a Kickstarter-exclusive functional groups table bookmark.
You'll be emailed the PDF and the handbook, thanked in the book, receive the physical book + 5) all of the original Photoshop files in the book, free for you to edit and use in your own presentations and handouts.
You'll be emailed the PDF and the handbook, thanked in the book, receive the physical book, all of the original Photoshop files, + 6) a classroom set of 25 books to use!


The conventional work triangle that divides the sink, stove and fridge has developed to a more functional "work zone" in today's world. The kitchen has become a much more versatile place and made to accommodate more people. Check out these different designs of kitchens that may make you want to remodel.
Initially Known as the "Pullman kitchen," that the one-wall kitchen design is usually located in studio or attic spaces since it is the ultimate space saver. Cabinets and appliances are fixed onto one wall. Most contemporary designs also incorporate an island, which evolves the distance to a type of Galley style using a walk-through corridor.
This efficient, "lean" design is excellent for smaller spaces and one-cook kitchens. The galley kitchen, also referred to as a walk-through kitchen, is distinguished by two partitions reverse of every other–or 2 parallel countertops using a walkway between them. Galleys create the best use of every square inch of distance, and there aren't any troublesome corner closets to configure, which may add into a cabinetry funding.
An L-shaped kitchen simplifies the problem of optimizing Corner distance, and it is a wise design for small and moderate sized kitchens. The flexible L-shaped kitchen is composed of countertops on two adjoining walls which are vertical, forming a L. The "legs" of the L might be as long as you need, however keeping them than 12 to 15 ft permit you to effectively use the distance.
Having an appearance design, you will eliminate traffic your kitchen won't develop into a thoroughfare since it's simply not logistically possible. Besides, it is simple to add a dining room and several work zones for this design. But, avoid this design if your kitchen is big and may encourage different configurations, like including an island, or when multiple cooks will probably use the space.
The horseshoe, or U-shape, kitchen design has three Walls of cabinets/appliances. Now, this layout has developed from three partitions into an modernized kitchen with an island forming the next "wall" "This layout works well since it allows for traffic circulation and workflow round the island," says Mary Jo Peterson, chief, Mary Jo Peterson Inc. "You may put more cooks into the kitchen".
A Functioning kitchen island could consist of cabinetry and appliances for storage and it constantly adds extra work surface into a kitchen. It can offer a place to eat (with stools), to prepare meals (using a sink) and to keep drinks (using a wine cooler). The island may turn a one-wall kitchen into a galley design, and also an L-shaped design into a horseshoe.
Kitchen islands are extremely Functional, however, the No. 1 misperception about islands is that everybody should own one. The fact is, many kitchens just don't have sufficient clearance to incorporate this attribute.
A peninsula kitchen is basically a island that is connected, Putting an L-shaped layout to a horseshoe, or turning into a horseshoe kitchen To a G-shaped design. Peninsulas work similar to islands but provide greater Clearance in kitchens which don't permit proper square footage to get a real island.


If you're like most people, there's probably an area in your life where you'd like to boost your confidence. And here's the good news… increasing one's confidence is much easier than you might imagine.
Most of us have tried all sorts of strategies to deal with the critical voices that erode our confidence. You know the ones that tell us that were not enough, that we're unworthy or undeserving. And often times we don't even hear a verbal message but are just flooded with the emotions of insecurity and fear.
and this Lifeforce is caught up in this program.
If your critical voices are really loud it can be quite difficult to access compassion to calm all this noise down. If you really want to be free quickly you may want to consider a private session.
In the meantime, be gentle with yourself, and breathe.


Details:As the temperature starts to drop and snow begins to fall bring the winter wonderland to your party by decorating with the Snowflake Party Panels! 12 inch X 6 feet. ; (3/Pkg); You get 12 packages for this price.


text:
Americans' low-carb attempts fall short
April 5, 2004, 10:48 PM UTC
By Jon Bonné
With a national obsession blossoming about low-carb options, have carbohydrates indeed been banished from the American diet?
Not a chance, says a leading food research firm. Even the most fastidious low-carb eaters are still consuming as much as twice as many carbs as recommended by popular diets.
"What you say and what you do are not always the same," said Harry Balzer, vice president of NPD Group.
The average American adult swallows 210 grams of refined carbs (that's total carbohydrates, minus dietary fiber, for those who've been buried under a pound of pasta) each day, according to NPD, which tracks U.S. eating habits. Even among the bottom 5 percent of carb consumers -– meaning 19 of every 20 people were eating more carbs than they were –- the average was still 145 grams for men and 109 grams for women, a combined average of 128 grams.
Low-carb diets like Atkins recommend as little as 20 grams of effective carbohydrates each day in early phases.
Certainly, though, the growing attraction of low-carb eating is apparent in NPD's findings, even if people aren't sticking to the specifics of details outlined by people like diet guru Dr. Robert Atkins.
Millions have tried
Some 17 percent of the 11,000 respondents in NPD's survey said they had tried low-carb eating at some point and 4 percent said they were currently on a high-protein, low-carb diet -– which would amount to some 10 million Americans. Research in 2003 by Harris Interactive for Novartis found that 32 million Americans were on a low-carb diet.
The Atkins' diet and many other low-carb plans have gained enormous appeal because they suggest you can eat plenty of foods once considered too fatty –- everything from red meat to rich cheese -– so long as you trim back on carbohydrates. It is a message that has truly resonated in a nation now saddled with an obesity epidemic, with 31 percent of adults considered obese and another third overweight.
But even among the 4 percent currently dieting, Balzer said, three out of four respondents were outside that bottom 5-percent group, meaning most were getting more than 35 percent of their calories from carbs.
"We tend to modify all the diets to reflect what we want to do," Balzer said. "This has given Americans a way of thinking about how they can lose weight while they still eat."
Atkins Nutritionals won't specify exact levels of net carb consumption for dieters who are trying to maintain their weight. But the company's nutritionists have suggested that from 60 to 120 grams of carbs would be appropriate long-term, depending on a consumer's health. The high end, which approaches levels found by NPD, might be acceptable for a younger, healthy dieter.
"This is an opportunity for us to help people refine the message," said Dr. Stuart Trager, chairman of the Atkins Physicians Council, which advises the company on its nutritional policies. "It's possible that many people are early on the learning curve."
'It's delivering'
But Balzer believes the low-carb craze is likely to be short-lived ("2004 is going to be the peak year for low carbs," he says) and soon to be replaced with another diet strategy. He likens it to the nation's obsession with low-fat options, which peaked in the 1990s before being replaced by a low-carb focus. That said, Balzer notes that food companies often require years to develop new products, which led to low-fat products on the shelves long after interest faded.
As such, the market for low-carb foods may be far broader than the number of people actually trying to cut back: Many of us may be chomping down on all those protein-rich, fiber-filled snack bars, but we're not truly committed.
Trager dismisses the notion that low-carb is a fad. He sees a bright future not just in the products but in long-lasting lifestyle changes voluntarily chosen by adherents of Atkins and other plans. "Low-fat was prescribed but didn't produce," he said. "Low-carb was discovered by people and it's delivering."
However, Trager noted, use of low-carb products –- especially snacks -– by people who do not follow broader diet guidelines is unlikely to do much for weight loss.
Those products may stick around for a while, Balzer believes, if their taste and the reputed benefits continue to appeal to consumers. But the low-fat years provided some costly lessons to food makers about the quirks of America's palate. Products like Nabisco's SnackWells continue to linger on shelves, but never matched dieters' zeal for less fat.
A fading fad?
Already, the low-carb craze is simultaneously expanding and inviting a backlash.
Atkins offers numerous books by its namesake founder, Dr. Robert Atkins, who died last year, in addition to a growing product line of low-carb foods and snacks. The South Beach Diet, essentially a lower-fat version of Atkins by Miami cardiologist Arthur Agatston, has garnered praise even from nutrition gadflies at the Center for Science in the Public Interest.
National chains like Subway and T.G.I. Friday's have offered low-carb entrées, "low-carb" food labels are manifest and hardly an industry segment –- even pizza -– has gone untouched. Low-carb food stores have sprung up from coast to coast.
But a sort of cultural counterpoint has ensued, most visibly when reports this past winter claimed Atkins was overweight and had heart disease when he died. (His company and relatives vigorously countered those claims.) And some nutritionists have assailed the Atkins plan for its tolerance of high-fat foods.
"If all of these claims that opponents make were true, people wouldn't be trying it," Trager said.
Perhaps the strangest of NPD's findings is who's eating the most carbs: adults who are at a proper weight or even underweight. By contrast, those eating fewer carbs report more health problems, high blood pressure and high cholesterol. And though 40 percent of low-carbers -– the majority of which are middle-aged -- say they exercise regularly, they're also 30 percent more likely to be obese.
It is, however, unclear which trend is leading to which: Are thinner people eating more carbs because they can (and fatter people eating fewer carbs because they're more inspired to lose weight) or are their diets determining their weight?
Balzer remains mystified. But he believes we may be reaching the end of our fondness for simple diet solutions, and are still consuming per capita as much as we ever have -- trying to modify our diet rather than simply eating less. It helps explain why the United States is consistently one of the top two nations in the world for calorie intake, at over 3,600 a day.
"If you really want to get Americans to eat healthy, the answer is very simple … tax food," he said. Then he hesitated.
"I don't like the word 'tax' … but you've got to make healthy foods less expensive."
Jon Bonné


With an adjustable temperature purification system that allows you to set the degree to your desire for your coffee and tea by tuning the knob.
A brand-new drinking water filtration system, integrating filtration and heating function featured in a compact size to provide better convenience.
Imported inner pleated membrane filter with MR FACT technology, pore size down to 0.2 micros with an advance ability to reduce sediments, chlorine, VOCs and other undesirable tastes in your water.
Safety lock to reduce risk of personal injury with manual deactivation upon dispensing hot water.
LED indicator for HEATING function that blinks during heating process and indicates high temperature after heating.
LED indicator for FILTER CHANGING function to alert for filter replacement.
LED indicator for LOW PRESSURE ALARM function to request user to check for water pressure.