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TFs (transcription factors) are modular proteins minimally containing a DBD (DNA-binding domain) and a TRD (transcription regulatory domain). NAC [for NAM (no apical meristem), ATAF, CUC (cup-shaped cotyledon)] proteins comprise one of the largest plant TF families. They are key regulators of stress perception and developmental programmes, and most share an N-terminal NAC domain. On the basis of analyses of gene expression data and the phylogeny of Arabidopsis thaliana NAC TFs we systematically decipher structural and functional specificities of the conserved NAC domains and the divergent C-termini. Nine of the ten NAC domains analysed bind a previously identified conserved DNA target sequence with a CGT[GA] core, although with different affinities. Likewise, all but one of the NAC proteins analysed is dependent on the C-terminal region for transactivational activity. In silico analyses show that the NAC TRDs contain group-specific sequence motifs and are characterized by a high degree of intrinsic disorder. Furthermore, ANAC019 was identified as a new positive regulator of ABA (abscisic acid) signalling, conferring ABA hypersensitivity when ectopically expressed in plants. Interestingly, ectopic expression of the ANAC019 DBD or TRD alone also resulted in ABA hypersensitivity. Expression of stress-responsive marker genes [COR47 (cold-responsive 47), RD29b (responsive-to-desiccation 29b) and ERD11 (early-responsive-to-dehydration 11)] were also induced by full-length and truncated ANAC019. Domain-swapping experiments were used to analyse the specificity of this function. Chimaeric proteins, where the NAC domain of ANAC019 was replaced with the analogous regions from other NAC TFs, also have the ability to positively regulate ABA signalling. In contrast, replacing the ANAC019 TRD with other TRDs abolished ANAC019-mediated ABA hypersensitivity. In conclusion, our results demonstrate that the biochemical and functional specificity of NAC TFs is associated with both the DBDs and the TRDs.
Gene-specific TFs (transcription factors) are DNA-binding regulatory proteins which activate or repress the basal transcription apparatus at target gene promoters. TFs are grouped into families on the basis of sequence similarities, most often in the DBD (DNA-binding domain) [1]. TFs from the same family often bind DNA in a sequence-specific manner, thereby only targeting promoters with a given consensus sequence [1]. Apart from the DBD, TFs are characterized by having a TRD (transcription regulatory domain). In contrast with the sequence similarities and well-defined structures of DBDs in members of a specific TF family, TRDs have traditionally been classified according to their amino acid profile, i.e. as acidic, glutamine-, proline- or serine/threonine-rich. Moreover, TRDs often have a high degree of low-complexity sequences and a propensity for flexible protein segments that fail to self-fold into an ordered three-dimensional structure, commonly referred to as ID (intrinsic disorder) [2].
Ongoing efforts to understand the structural and functional modularity of TFs have traditionally been based on the examination of the activity of truncated versions of the proteins; an early discovery was that the DBD could be separated from the TRD with no loss of function of either module which is illustrated by the yeast GAL4 TF, a paradigm for eukaryotic transcriptional regulation studies [3]. Additionally, Johnston et al. [3] showed that expression of a cytoplasmically located version of GAL4, without the N-terminal DBD, in a wild-type background enabled induction of the expression of reporter genes to the same extent as expression of full-length GAL4. This implied that GAL4 consists of separate domains conferring nuclear localization, specific protein–protein interactions, DNA-binding and transcriptional activation [3]. This seemingly independent modularity has proven to be a general model for many TFs and has led to the question as to how functional specificity is substantiated [4,5].
Although a wealth of studies have aimed to characterize TF functionality, a few of them can be highlighted as important attempts to shed light on functional specificity from analysis of independent TF modules. In yeast, this question has been addressed by deciphering the functions of regions within the transcriptional activator Rap1p (repressor activator protein 1), showing that the C-terminal TRD has only a minor effect on the cell growth effect modulated by Rap1p [6]. Furthermore, domain-swapping experiments with combinations of elements from DBDs of Rap1p homologues from different yeasts revealed that major changes can be made to the amino acid sequence of this region without affecting the telomere-binding function of Rap1p [6]. In another study, using a series of domain-swap chimaeras in which different parts of Drosophila melanogastor T-domain TFs, encoded by the genes omb (optomotor-blind) and org-1 (optomotor-blind related-1), were mutually exchanged, Porsch et al. [7] investigated the relevance of individual domains for proper eye development. Their findings suggested that both transcriptional activation and repression properties, as well as the DNA-binding specificity, can contribute to the functional characteristics of T-domain factors [7], highlighting the complexity of parsing the functional specificity of seemingly independent TF domains.
In plants, the NAC [for NAM (no apical meristem), ATAF, CUC (cup-shaped cotyledon)] family constitutes a prominent group of TFs [8]. The genomes of Arabidopsis thaliana (thale cress), tobacco and rice all contain more than 100 genes encoding NAC TFs, making it one of the largest gene families in plants [9,10]. Genes encoding NAC TFs were originally identified from forward genetic screens as key regulators of developmental processes [8]. More recently, NAC TFs have also been shown to be involved in the regulation of stress responses in both model plants and agronomically important crops [11,12].
We have shown previously the NAC protein ANAC019 interacts with the RING-finger H2-type E3 ubiquitin-protein ligase RHA2a, and we have solved the crystal structure of the ANAC019 NAC domain [13,14]. The structure revealed a novel protein fold consisting of a twisted β-sheet packing against an α-helix on both sides. Moreover, using ANAC019 as a reference we elucidated residues important for both NAC homo- and hetero-dimerization and for binding to a core consensus DNA element of sequence CGT[GA] [15]. Studies of ANAC019 gene expression have implicated a role in different types of stress responses [14,16] and overexpression of ANAC019 caused increased drought tolerance [16]. Moreover, it has been shown that ANAC019 regulates defence responses upon fungal attack through a signal pathway related to the plant hormone jasmonic acid [17]. Recently, Bu et al. [18] verified the ANAC019–RHA2a interaction and defined RHA2a as a new positive regulator of ABA (abscisic acid) phytohormone signalling [18].
With regard to parsing functional determinants of NAC DBDs and TRDs, Taoka et al. [19] have shown that for members of the CUC subfamily of NAC TFs the DBD confers functional specificity in terms of inducing adventitious shoot formation on calli [19,20]. TRDs from three different NAC TF origins could be fused to the NAC domain of CUC2 and still induce shoot formations. In contrast, replacing the CUC NAC domains with a NAC domain from a member of the ATAF subfamily of NAC TFs did not induce shoot formation. However, the NAC domain of the CUC subfamily member CUC1 did not retain shoot-formation-ability when fused to the potent TRD from the herpes simplex virus VP16 protein. This suggested that certain structural features of the TRD, which are difficult to predict from the amino acid sequence, were necessary for the function of CUC [19].
In the present study, we have used a genome-wide approach, taking advantage of the functional diversity of the NAC proteins, to dissect structure–function aspects of NAC TF modularity. Our analyses showed that phylogenetic relationships based on NAC domain sequences are generally in accordance with the C-terminal sequence motifs of the intrinsically disordered TRDs and global NAC gene expression clusters. Additionally, our study includes examination of truncated versions of ten phylogenetically distinct NAC proteins to characterize the structural specificity of NAC DBDs and TRDs. We also use a chimaeric strategy, based on our identification of ANAC019 as a novel ABA regulator, to investigate the structure–function relationship of the ANAC019 DBD and TRD. The findings suggested multi-specificity of NAC TFs, in terms of the DNA-binding sites in target genes, and provided evidence that positive ABA-regulatory functionality is associated with both the ANAC019 TRD and DBD.
Oligonucleotides and restriction enzymes
Oligonucleotides and restriction enzymes used in the present study are listed in Supplementary Table S1 (available at http://www.BiochemJ.org/bj/426/bj4260183add.htm). The DNA sequence integrity of all expression constructs was confirmed by sequencing.
Accession numbers
Sequence data from this article can be found in TAIR (The Arabidopsis Information Resource) and EMBL (European Molecular Biology Laboratory) data libraries under the following NAC nomenclature names and accession numbers: ANAC019, At1g52890; ANAC003, At1g02220; ANAC002 (ATAF1), At1g01720; VOZ2 (vascular plant one-zinc finger protein 2); At2g42400; ANAC008 [(SOG1 (suppressor of gamma response 1)], At1g25580; ANAC105 [VND3 (vascular-related NAC domain 3)], At5g66300; ANAC030 (VND7), At1g71930; ANAC092 (NAC2/ORE1), At5g39610; ANAC062 (NTL6), At3g49530; and ANAC040 (NTL8), At2g27300.
Bioinformatic analysis of the NAC family
BLASTP and PSI (position-specific iterated)-BLAST searches were performed using the NCBI (National Center for Biotechnology Information) BLAST services. Searches were performed against a non-redundant database, but were limited to include only hits within the Arabidopsis genome. All sequences were annotated according to the latest Arabidopsis genome annotation. Multiple alignments were generated using ClustalW or ClustalX [21] and BoxShade (http://www.ch.embnet.org/software/BOX_form.html) was then used for producing graphical presentations of the alignments and for manual adjustments. Phylogenetic analysis of the NAC domain sequences was performed using MEGA4 software [22]. The input was a multiple alignment of NAC domain sequences generated in ClustalX. The reliability of the different groupings of the tree was measured by bootstrapping. MEME [multiple EM (expectation maximization) for motif elicitation] was used to search for statistically significant motifs in the NAC proteins using either all NAC sequences or group- and subgroup-specific sequences as a data set. MAST (motif alignment and search tool) was used for further analysis of the NAC proteins. P values were used to determine the overall match of the sequence to the input motif. In addition, the relevance of the motifs identified was evaluated by analysis of its occurrence in control groups, e.g. the NAC domain served as a control group for motifs identified in the C-terminal regions and vice versa. Amino acid profiling was performed using Composition Profiler [23] taking advantage of the standard amino acid data set, SwissProt51, provided by the program. PONDR VL3 [24] was used for prediction of structural ID.
For global expression analysis, normalized At-TAX (tiling array express) expression data for 107 NAC genes during selected developmental stages and in response to different environmental stresses was obtained [25]. Expression values were imported into the R-software for processing and heatmap output using the Bioconductor microarray analysis packages affy, heatplus and gplots [26].
Plant materials, growth conditions and Arabidopsis transformation
Arabidopsis Col-0 (Columbia ecotype 0) wild-type, the mutant homozygotes anac019 (SALK_096303) and transgenic seeds were grown in growth chambers at 22 °C and with light intensity of 150 μmol/m2 per s from a combination of fluorescent and incandescent lamps. Transformation of Arabidopsis Col-0 plants was performed by a modified floral dip infiltration method [27] using the Agrobacterium tumefaciens strain GV3101 (pMP90) and the binary vector pCAMBIA3300 (Cambia) containing the CaMV (cauliflower mosaic virus) 35S promoter for constituent expression of transgenes and the BAR selectable marker conferring tolerance to the herbicide Basta (50 mg/l glufosinate ammonium). Transgenic plants were selected by Basta spraying and T3 seeds from transformants expressing transgenes were used for subsequent analyses. An empty pCAMBIA3300 vector was transformed into Col-0 wild-type plants as a control.
ABA sensitivity assay
To determine the effect of ABA on germination and post-germination growth efficiency, surface-sterilized and stratified (3 days) seeds of Arabidopsis Col-0 wild-type, anac019 mutant alleles (SALK_096303) and transgenic lines expressing full-length, truncated or chimaeric constructs of ANAC019, were grown on half-strength Murashige and Skoog medium supplemented with 0–5 μM ABA (mixed isomers; Sigma–Aldrich). A seed was considered as germinated when the radicle penetrated the seed coat. The percentage of germinated seeds was scored at 3 days after stratification in three independent experiments (40 seeds/experiment per treatment). Post-germination growth efficiency was scored as primary root length at 7 days after stratification. Seeds from Col-0 wild-type, anac019 mutants and transgenic lines used for phenotypic analysis were harvested and collected at the same time. Root elongation assays were performed as described in [28] and significance levels were assessed by a two-sided t-test.
Production of recombinant proteins and in planta expression
For production of recombinant NAC proteins, cDNA clones obtained from the ABRC (Arabidopsis Biological Resource Center) and were amplified by PCR to obtain the complete coding sequence or the region encoding the NAC domain of ANAC019, ATAF1, NAC2/ORE1, VOZ2, VND3, VND7, NTL8, SOG1, NTL6 and ANAC003 using the primer sets listed in Supplementary Table S1. The PCR products were inserted into pENTR/D-TOPO (Invitrogen) followed by recombination into pDEST-15 (Invitrogen) to obtain GST (glutathione transferease)-tagged NAC recombinant proteins. The recombinant proteins were named to show the borders of the NAC domain fragment, e.g. GST–ANAC019-(1–168), which consists of GST fused at the N-terminus of the 168 N-terminal residues of ANAC019. Escherichia coli strain BL21(DE3) was transformed with the expression constructs and used for expression of GST–NAC recombinant proteins. Transformed E. coli cultures were grown to a cell density (D600) of 0.6 in Luria–Bertani medium before induction with 0.5 mM IPTG (isopropyl β-D-thiogalactoside). After 3 h of incubation at 37 °C the cells were harvested by centrifugation at 10000 g for 20 min. The cell pellet was resuspended in PBS, pH 7.5, containing 1 mM DTT (dithiothreitol), and was incubated on ice for 30 min before sonication using a Soniprep 150 instrument (MSE) at amplitude 7 μ for 30 s, pausing for 1 min, and then repeating the process three times. Following sonication, the lysate was cleared by centrifugation at 10000 g for 20 min. The GST–NAC fusion proteins were purified by affinity chromatography using a 2 ml glutathione–Sepharose 4B column (GE Healthcare) with 250 ml of E. coli culture (at the time of harvest D600 was approx. 3.0–4.0). The column was washed with ten column volumes of PBS, pH 7.5, containing 1 mM DTT. The protein was eluted in 1-ml fractions in a buffer of 50 mM Tris/HCl, pH 8.0, containing 10 mM GSH. If required, the proteins were purified further by anion-exchange chromatography. The protein solutions were dialysed overnight against a buffer of 20 mM Tris/HCl, pH 8.0, containing 1 mM DTT and loaded on to a 1-ml RESOURCE Q column (GE Healthcare) and eluted using a gradient of NaCl.
The vector pCAMBIA3300 and the primers and restriction enzymes listed in Supplementary Table S1 were used for generation of chimaeric constructs for in planta expression. Full-length ANAC019 (residues 1–317) was amplified using a cDNA clone obtained from the ABRC. Chimaeric constructs for domain-swapping experiments that contained the ANAC019 NAC domain and divergent TRDs were constructed. First, the DNA fragment corresponding to the ANAC019 NAC domain (residues 1–168 of ANAC019) was PCR amplified using a full-length cDNA clone from ABRC; for overlap extension PCR with fragments encoding the ANAC019 NAC domain, the reverse primers included a 12-bp extension at their 5′-termini. Then DNA fragments encoding the TRDs [NAC2/ORE1-(175–285), NTL8-(158–335) and SOG1-(216–449)] were PCR amplified using cDNA clones from ABRC; for overlap extension PCR, forward primers for DNA amplification of sequences encoding TRDs, included a 12-bp extension in their 5′-termini. Upon successful generation of the individual fragments, the two amplification products were mixed and amplified using the following PCR program: four cycles of 94 °C for 3 min, 53 °C for 30 s and 72 °C for 3 min. The PCR was performed with 2.5 units of Pfu DNA polymerase (Fermentas), 0.2 mM dNTPs and approx. 150 ng of individual DNA fragments in a 100 μl reaction volume. Immediately afterwards, 0.3 μM external primers were added and the constructed insert was amplified using the following PCR program: 95 °C for 2 min; 30 cycles of 94 °C for 1 min, 52 °C for 1 min, 72 °C for 2 min; 72 °C of 15 min; and holding at 4 °C. Chimaeric constructs that contained the ANAC019 TRD and divergent NAC domains were constructed as described above, amplifying the NAC domains instead of the TRDs [NAC2/ORE1-(1–174), NTL8-(1–157) and SOG1-(1–215)], and the TRD of ANAC019 (residues 169–317) instead of the ANAC019 NAC domain.
EMSA (electrophoretic mobility-shift assay)
A double-stranded oligonucleotide containing an optimized palindromic version of the NAC-BS (binding site) selected by CASTing (cyclic amplification and selection of targets) [15] was prepared by annealing complementary single-stranded oligonucleotides of the sequence 5′-CAGTCTTGCGTGTTGGAACACGCAACAGTC-3′. The double-stranded oligonucleotide was labelled at the 3′ end with DIG (digoxigenin) using a DIG gel-shift kit (second generation; Roche). DNA binding reactions contained 4 μl of 5× binding buffer {100 mM Hepes, pH 7.6, containing 5 mM EDTA, 50 mM (NH4)2SO4, 5 mM DTT, 1% (w/v) Tween-20, 150 mM KCl, 1 μg of poly[d(I-C)]}, 31 fmol of DIG-labelled DNA and the indicated amount of GST–NAC domain protein in a final volume of 20 μl. The binding reactions were incubated for 15 min at room temperature (20 °C) and separated by PAGE (10% PAGE gold precast gels; Cambrex). Electroblotting was performed using a Hybond-N nylon membrane (Amersham Biosciences) and chemiluminescent detection was carried out according to the manufacturer's instructions.
Transactivation analysis in yeast
NAC proteins were examined for the presence of an activation domain using a yeast-based reporter assay. Full-length or NAC-domain-truncated coding sequences of the ten NAC candidates were cloned into the DBD vector pGBKT7 (Clontech). Primers and restriction enzymes used are listed in Supplementary Table S1. Subsequently, constructs encoding the GAL4 DBD fused to full-length or NAC-domain-truncated versions of NAC protein coding sequences were transformed into the yeast strain pJ694A, which harbours the HIS3 and ADE2 reporter genes. The transformed yeast cultures were plated on to SD (synthetic defined) plates without tryptophan (−Trp) and SD plates without tryptophan, histidine and adenosine (−Trp/−His/−Ade) for 7 days at 30 °C before inspection of transactivation properties of the reporter constructs. For easy assessment of transactivational activity, overnight cultures of transformants were diluted to a D600 of 0.6, and then 10-fold serial dilutions were prepared. Finally, 5 μl of each dilution was spotted on to SD −Trp plates and SD −Trp/−His/−Ade plates, which were then incubated at 30 °C for another 7 days. Constructs pVA3–1 (containing the p53 DBD) and pTD1–1 (containing the T-antigen activation domain) were used as positive controls. Yeast cells transformed with pGBKT7 vector alone cannot activate the GAL1 promoter and thus served as a negative control.
RNA extraction, cDNA synthesis and quantitative real-time RT (reverse transcription)–PCR analysis
For Arabidopsis transcript analysis, total RNA was isolated from three complete rosettes for each genotype, using the RNeasy mini kit (Qiagen). Purified DNase-treated RNA (1 μg; Promega) was used for cDNA synthesis using the Superscript III cDNA synthesis kit (Invitrogen) according to the manufacturer's instructions. Quantitative real-time PCRs were performed in triplicate for each individual line, and quantification of CT (cycle threshold) values was achieved by calculating means of normalized expression using Q-gene software [29]. Actin2 was used as a reference to determine the relative expression of the ABA-responsive genes COR47 (cold-responsive 47; At1g20440) and RD29b (responsive-to-desiccation 29b; At5g52300), and the stress-responsive genes FER1 (ferritin precursor 1; At5g01600) and ERD11 (early-responsive-to-dehydration 11; At1g02930). Experiments were repeated three times with similar results. Primers for quantitative real-time PCR are listed in Supplementary Table S1.
The Arabidopsis NAC family
To uncover the expansion of the Arabidopsis NAC TF family we searched for NAC sequences using BLASTP in latest genome annotation, the translated Arabidopsis genome, using the NAC domain of ANAC019 [14] as a query. This approach identified 92 non-redundant Arabidopsis proteins, and 18 additional NAC proteins were uncovered in an iterative PSI-BLAST search. A multiple ClustalX alignment of the 110 sequences revealed one sequence with only partial similarity to the NAC domain consensus, leaving 109 NAC sequences (see Supplementary Table S2 available at http://www.BiochemJ.org/bj/426/bj4260183add.htm). To study the Arabidopsis NAC TF family in more detail, a phylogenetic tree was constructed with the conserved NAC domains in MEGA4 [22], using a neighbour-joining method and bootstrap analysis to assess branch reliability (Figure 1). As MEGA4 was unable to construct a tree based on alignments including At1g60240, this sequence was left out. The phylogeny of the 108 Arabidopsis NAC is presented in Figure 1 and an alignment of selected NAC domains is presented in Figure 2(A). On the basis of the phylogenetic analysis, the Arabidopsis NAC family was divided into ten major polytomies by collapsing branches with bootstraps <40%. Aside from the ten major groupings, several minor groupings were observed. To keep the Arabidopsis NAC nomenclature consistent, names from previous nomenclature systems [9,30] are maintained when possible in this updated nomenclature (see also Supplementary Table S2).
Phylogenetic and expression analyses of the Arabidopsis NAC family
Structural features of NAC proteins
Global NAC gene expression analysis
Knowledge of NAC gene expression patterns has contributed significantly to our understanding of NAC gene involvement in plant stress tolerance and development [25]. To provide a global overview of the NAC transcriptome in relation to different developmental stages and environmental stresses we analysed the Arabidopsis gene expression data accessible online at the At-TAX website (http://www.weigelworld.org/resources/microarray/at-tax) [25]. In contrast with the limited number of NAC gene probe sets available from the widely used ATH1 GeneChip, At-TAX expression data is available for almost all of the 108 NAC genes listed in Figure 1 (locus At1g64105 overlaps At1g64100 and has no unique probes), and therefore provided the basis for the expression analysis.
The At-TAX expression profiles showed that, overall, NAC gene expression is detected in all selected tissues and developmental stages (Figure 1). Likewise, NAC transcript levels are widely affected by a range of environmental stresses. Additionally, without posing any clustering of the expression data, minor groupings with similar expression patterns correlate with the phylogenetic analysis based upon the NAC domain sequences. This is particularly evident for the tissue-specific expression patterns, e.g. a fraction of the VND subgroup (II-1) of NAC genes are predominantly expressed in both stems and roots, whereas others are expressed only in roots or stems. These data support previous studies of VND members which identified them as important regulators in transdifferentiation of various cells into vessel elements [31,32]. Another prominent subgroup is the ATAF (III-3) group, which shows the most abundant co-expression levels in old leaves and roots. This is also the case for the more distantly related subfamily VII-2 and for members of the transmembrane-anchored subgroups I-1 and I-2. Moreover, gene members of subgroup III-3 are highly salt-inducible, whereas those of subfamily I-1 and VII-2 mainly respond to elevated temperatures. Finally, the co-expression of the five NAC genes encoding two NAC domains (VIII-1) in whole inflorescence and fruits deserves to be highlighted as a yet-to-be characterized subgroup with a distinct co-expression pattern correlating with phylogenetic distribution. Overall, among the displayed stress data sets, factors affecting the expression of most NAC genes are salt and extreme temperatures. With respect to the latter, most NAC genes shown to be induced by cold are repressed by heat and vice versa. In conclusion, several phylogenetically related NAC genes are co-expressed in a developmental/tissue-specific, and to a lesser extent stress-specific, manner.
Structural characterization and classification of the NAC proteins
To further analyse the structural diversity of Arabidopsis NAC proteins, sequence motifs were predicted using the programs MEME and MAST. This identified both shared and group-specific sequence motifs in the NAC domains (Figures 2A and 2B; Supplementary Table S2). The major motifs mapped to the α-helices and β-strands of the NAC domain [13]. Whereas most of the NAC domains contained motifs we designated as A–F, domains from groups VI–X shared fewer motifs with the rest of the NAC domains, and some had subgroup specific motifs, such as motif R of subgroup IX-1 (Figure 2B).
The divergent C-terminal regions of NAC proteins, for which no tertiary structure information is available, were also examined for sequence motifs (Figure 2B and Supplementary Table S2). Subgroup II-1 of NST/VND proteins shared motifs WQ and LP, of which WQ has been shown to be important for transactivational activity [33]. Three motifs, LP (also named L; [19]), V and W, dominate the C-termini of the CUC-like proteins, which includes NAC2/ORE1 [34], of subgroup II-3. Motif W was reported to be of importance for transcriptional activity [19,33]. Thus most of the C-termini of group II proteins share motif LP and probably depend on a motif with a prominent tryptophan for activity (Figure 2B). Additional motifs, such as low-complexity histidine- and glutamine-rich Q, U and T motifs were identified in some group II proteins. Group I includes most of the fourteen NAC proteins with a C-terminal transmembrane region [8,35]. For these proteins, the majority of the C-terminal motifs (e.g. l, Y, b, K and k) are dominated by polar and negatively charged residues with conserved hydrophobic residues embedded in the polar matrix (e.g. isoleucine and leucine residues in motif l and tryptophan and phenylalanine residues in motif K). Subgroup III-3 contains the stress-related proteins, ANAC019, ANAC055, ANAC072, ATAF1 and ATAF2, which also have motifs found only in their subgroup (o, g, h, n and i). These motifs, and motif O of subgroup V-II, are also dominated by a negatively charged matrix with a few conserved bulky and hydrophobic amino acid residues (Figure 2B; Supplementary Table S2).
The groups discussed above represent the most highly studied NAC proteins, and comply with the classical modular outline presented in Figure 2(C). In contrast, the proteins in subgroup VIII-1 consist of two tandemly repeated NAC domains (Figure 2C) and have genes without annotated introns. Subgroup IX-1 forms a distinct clade, as supported by high bootstrap values, and contains the recently characterized SOG1 gene [12]. The seven members of this subgroup deviate from the characteristic NAC structure by having N-terminal extensions of approx. 40 amino acid residues with a pair of conserved cysteine residues (motif R; Figures 2B and 2C). Some of these proteins also have typical, but specific C-terminal motifs (motifs d and e). In the phylogenetic tree, subgroup IX-1 is closely related to the VOZ proteins (subgroup VIII-2 [36]), which were identified as NAC proteins in PSI-BLAST searches [15]. The VOZ proteins have an N-terminal region of approx. 240 residues followed by a conserved DBD, comprising of a zinc co-ordinating motif and a C-terminal basic region with structural similarity to the NAC domain (Figures 2A and 2C). In the N-terminal domain, the VOZ proteins contain motifs a and d which are chemically similar to characteristic C-terminal NAC motifs. In conclusion, some subgroups have specific motifs in the NAC domain, reflecting their divergence from the core NAC domain. Moreover, C-termini of NAC proteins contain several group-specific motifs, which are enriched in polar and acidic residues.
Selection of representative NAC proteins
Most studies of NAC family members have focused on members from groups I–III, which have typical NAC domains that bind DNA and a C-terminal TRD (Figure 2C) [15,16,19]. To understand the modular structures of NAC TFs with respect to functionality, ten NAC proteins, representing functionally important clades and spanning the phylogenetic diversity of the NAC family (Figure 1), were selected for characterization. On the basis of the extensive characterization of ANAC019 [13,15–17], we decided to use this protein as a reference in the comparative studies. The remaining nine NAC proteins were ATAF1, VND3, VND7, ANAC003, NAC2/ORE1, SOG1, VOZ2, NTL6 and NTL8 (Figures 1, 2A and 2B).
In vitro DNA-binding of structurally variant NAC domains
In our previous study we determined a consensus NAC-BS with the core sequence CGT[GA] using a reiterative selection procedure on random oligonucleotides and ANAC019 and NAC2/ORE1 as baits [15]. A CGT[GA] or CGT core consensus sequence has also been identified as a DNA-binding sites for other NAC TFs from both Arabidopsis and wheat [15,16,37]. To examine the in vitro DNA-binding properties of selected NAC proteins, N-terminally GST-tagged NAC domains were produced heterologously. All proteins were purified by affinity chromatography and some were purified further by ion-exchange chromatography. The quality of the recombinant proteins was examined by SDS/PAGE. The GST–NAC domain fusion proteins had the expected molecular mass values (of between 47 and 54.1 kDa) and only minor contaminants (Figure 3A).
Analysis of NAC domain DNA binding using EMSAs
Using the consensus NAC-BS, a palindromic NAC-BS, palNAC-BS, was designed for EMSA-based analysis of the DNA-binding of the NAC domain (Figure 3B) [15]. The palNAC-BS was used to examine the ability of GST–NAC proteins to bind DNA in a titration series using increasing amounts (0–500 ng) of GST–NAC with GST alone as control (Figure 3C). Preliminary experiments showed that GST–NAC domain proteins bound palNAC-BS with the same affinity as the corresponding NAC domain without a GST tag and that full-length NAC2/ORE1 bound palNAC-BS with the same affinity as the NAC2/ORE1 NAC domain (results not shown). For each protein a minimum of three EMSAs were performed, each time with an independent protein batch, and these all showed the same binding pattern. As expected, GST–ANAC019-(1–168) and GST–NAC2/ORE1-(1–165) bound palNAC-BS, and binding could be detected using 5 ng of the recombinant proteins. GST–ATAF1-(1–165) also bound palNAC-BS, with an affinity similar to that of ANAC019 (measured as the amount of protein needed to produced a shifted band). This is in accordance with the close relationship between ANAC019 and ATAF1 (Figure 1). In addition to the major band formed in the presence of GST–NAC and palNAC-BS, we sometimes observed a minor band, most often of slower migration (Figure 3C). This may represent non-native protein–protein interactions and has been observed in studies of other DNA-binding proteins [38]. A similar binding pattern was also seen for GST–VND7-(1–163), whereas the closely related GST–VND3-(1–165) bound with lower affinity. To our knowledge, the in vitro DNA-binding properties of proteins from the well-studied subgroup II-1 of VND proteins have not been analysed previously. GST–VOZ2-(205–420) also bound palNAC-BS, although with lower affinity than GST–ANAC019-(1–168), which was also the case for both GST–NTL8-(1–157) and GST–NTL6-(1–168).
So far neither ANAC003 nor SOG1, or members of their clades, have been characterized with respect to their biochemical properties and their sequences show a relatively low degree of similarity to the typical NAC domains. They both have substitutions in some of the positions corresponding to the residues Lys-79, Arg-85 and Arg-88 in ANAC019 (Figure 2A), which are of known importance to DNA-binding [15]. GST–SOG1-(1–220) showed a weak affinity for palNAC-BS. In contrast, attempts to show binding of GST–ANAC003-(1–152) to palNAC-BS in EMSAs were unsuccessful. In addition, the recombinant protein has no detectable binding to a 30-bp fragment of the CaMV 35S promoter (positions −84 to −55), which is bound by several NAC proteins (results not shown) [39,40], and attempts to select binding sequences for GST–ANAC003-(1–152) using CASTing were also unsuccessful. In conclusion, nine of the ten NAC domains examined bound palNAC-BS, although with differing affinities which generally reflected their structural relations. No DNA-binding ability was detected for ANAC003.
C-terminal regions of NAC proteins are intrinsically disordered
The C-terminal regions of NAC TFs were analysed further using the Composition Profiler software [23], which analyses the amino acid composition of a protein. This showed an under-representation of hydrophobic and structure-stabilizing amino acid residues and an over-representation of polar and negatively charged residues in the C-termini compared with the frequency in the reference dataset, SwissProt51 (Figure 4A). The only frequent basic residue in the C-termini is histidine as reflected by the histidine-rich motifs Q and U (Figure 2B). The N-terminal regions of the VOZ proteins have similar profiles (see Figure 2C for a schematic comparison of the canonical NAC modularity against the VOZ modularity). In contrast, the NAC domains have a higher frequency of basic and aromatic residues (Figure 4A).
Structural characteristics and predictions of NAC domains and adjacent regions
The compositional profile of the NAC TF C-termini is suggestive of ID [2]. For this reason, all NAC proteins were examined for disorder using the predictor PONDR VL3 [24]. This analysis confirmed our assumptions and showed that the C-termini have a large degree of ID, and that they were more highly disordered than the NAC domains (Figure 4B). The analysis was supported by a circular dichroism analysis of the C-terminal region of a barley NAC protein indicating that the region is largely unfolded (T. Kjaersgaard and K. Skriver, unpublished work). For some proteins, a subgroup-specific conserved motif is located in a dip in the disorder prediction, as exemplified by motif l of the subgroup I-4 proteins, motif i of some subgroup III-3 proteins and motif WQ of the VND subgroup II-1 proteins. In other cases, the motif is located in similar positions in the border between ordered and disordered regions (e.g. motif LP of subgroup II-3, motif K of subgroup I-2 and motif N of subgroup VII-2 proteins) (Figure 4B and results not shown). Such motifs may represent MoRFs (molecular recognition features) [41] with a propensity for target-induced folding. In conclusion, in silico analysis of the NAC protein C-termini showed that they are enriched in polar and acidic residues and have a large degree of ID.
Divergent NAC transcription regulatory domains
In several cases NAC proteins have been reported to have a C-terminal TRD [5,42]. This correlates with the high degree of ID in the NAC C-termini (Figure 4B), which is characteristic of TRDs [2]. To provide a systematic characterization of NAC TRDs we examined the transactivational activity of full-length and N-terminally truncated versions of the candidate NAC proteins. For this purpose fragments encoding full-length and truncated versions of the ten NAC proteins were fused in-frame to the GAL4 DBD in the yeast expression vector pGBKT7. The GAL4-(DBD)–NAC-(TRD) fusion proteins were subsequently expressed in a yeast strain containing the HIS3 and ADE2 reporter genes.
The transactivation analysis of full-length and truncated versions of ANAC019 confirmed our previous findings, identifying the C-terminal of ANAC019 as harbouring a transactivation domain (Figure 5A) [14]. However, and in accordance with Bu et al. [17], full-length ANAC019 did not show any transactivation potential in this assay, though Tran et al. [16] have shown that ANAC019 transactivates expression in a plant-based reporter assay. Interestingly, removing the extreme N-terminal region, ANAC019-(1–40), improved the transactivational potential (Figure 5A), a result reminiscent of NAC1 transactivational activity [5]. Taken together, these results showed that the ANAC019 C-terminal region has transactivational activity in yeast.
NAC transcription factors are transcriptional activators
Secondly, full-length VND7, NTL8, ATAF1 and VOZ2 have all been shown to activate transcription in yeast or plant-based reporter assays [32,36,43,44]. Furthermore, it has been shown that VND3 activates transcription through heterodimerization with VND7 [32], and that the TRDs of NTL8 and ATAF1 are located in the C-terminal part of the protein [43,44]. The reporter gene assay in the present study corroborates the findings on the full-length and C-terminal constructs analysed previously, with the exception of full-length NTL8 where the construct did not show transactivation, in contrast with observations by Kim et al. [43] (Figure 5B). However, apart from ANAC019 and NTL8, all full-length constructs were able to transactivate expression of the HIS3 and ADE2 reporter genes. Additionally, all the N-terminal truncation constructs analysed showed transactivational activity, apart from the non-canonical NAC transcription factor VOZ2, which was dependent on its N-terminal to confer transactivation (Figure 5B). This protein has its transactivating activity in the N-terminus (see Figure 2C). In conclusion, NAC domains are dispensable for conferring a transactivational potential, and the TRD usually resides in the C-terminal part of the protein.
ANAC019 regulates ABA signalling during seedling development
Expression of the ANAC019 gene is induced by drought, salt and ABA (Figure 1; [14,16]). ABA plays a key role in seed germination and early seedling development, as well as during plant drought and salt-stress perception [45]. To investigate the functional relationship between ABA and ANAC019, the ABA-dose response of Col-0 wild-type plants was compared with that of transgenic plants overexpressing full-length ANAC019-(1–317), ANAC019-(169–317) or ANAC019-(1–168) and to anac019 mutant plants using quantitative bioassays of germination (radicle emergence) and seedling development (Figures 6A and 6B). Ectopic expression of transgenes was verified using quantitative real-time RT–PCR (see Supplementary Figure S1 available at http://www.BiochemJ.org/bj/426/bj4260183add.htm). In the absence of exogenous ABA, all genotypes showed similar germination by day 3, except anac019 mutant plants which showed a significant decrease in the ability to germinate (P<0.05) (Figure 6A). In the presence of low ABA concentrations ANAC019-(1–317) and ANAC019-(1–168) showed lower germination efficiency compared with wild-type and anac019 mutant plants. At 2 μM ABA plants expressing either full-length or truncated versions of ANAC019 displayed ABA hypersensitivity compared with wild-type and especially anac019 mutant plants, and at 5 μM ABA only anac019 mutant seeds germinated (Figure 6A). During early seedling development, primary root length was scored at 7 days after stratification in the absence or presence of different concentrations of ABA (Figure 6B). At low ABA concentrations, plants expressing either ANAC019-(1–317) or ANAC019-(1–168) were more sensitive than the wild-type and anac019 mutant plants, as shown by shortened primary root elongation (0.05<P<0.001). At 2 μM ABA, plants expressing ANAC019-(1–317) and ANAC019-(169–317), but not ANAC019-(1–168), were significantly hypersensitive to ABA compared with wild-type and anac019 mutant plants (P<0.01). In summary, ANAC019-(1–317) and truncated versions of ANAC019 displayed ABA hypersensitivity during germination and early seedling development, suggesting that ANAC019 is a positive regulator of ABA signalling.
ANAC019 is a positive regulator of ABA signalling
To further understand the molecular basis of ANAC019-mediated ABA hypersensitivity, the expression profile of several ABA- and stress-responsive genes (RD29b, COR47, FER1 and ERD11 [16]) were investigated by real-time PCR (Figure 6C). Three out of the four genes analysed displayed increased transcript levels in plants expressing ANAC019-(1–317), ANAC019-(1–168) and ANAC019-(169–317) compared with control and anac019 mutant plants. The induction of FER1 expression was dependent on the C-terminal TRD of ANAC019 (Figures 6C and 7). Taken together, the up-regulation of the ABA-responsive marker genes in plants expressing full-length or truncated versions of ANAC019 largely correlated with the increased ABA sensitivity of ectopically expressing ANAC019 plants. The presence of a NAC-BS in the 1-kb promoter of all four genes (results not shown) highlights that ANAC019 is a possible direct positive upstream regulator of ABA-signalling genes.
Analysis of ANAC019-mediated ABA sensitivity by domain-swap analysis
Modular interdependency of ANAC019-mediated ABA signalling
Taoka et al. [19] showed that among close homologues of the CUC proteins of the NAC family, functional specificity resides in the N-terminal NAC domain. To examine if ANAC019-mediated perturbation of ABA-sensitivity can be mapped to specific domains, six chimaeric constructs were generated in which NAC domains of NAC2/ORE1, NTL8 and SOG1 were individually fused to the ANAC019 TRD and the TRDs of NAC2/ORE1, NTL8 and SOG1 were fused to the ANAC019 NAC domain (Figure 7A). All constructs were transformed into Col-0 wild-type plants. Selection of these candidate fusion partners enabled analysis of both distant and close homologues of ANAC019, which have both overlapping and non-overlapping expression profiles (see Figure 1). The effect of ectopic expression of these constructs was examined by scoring root length in the absence or presence of 2 μM ABA (Figure 7). In addition to conferring reduced root length in plants expressing ANAC019, this ABA concentration has also been shown to compromise root length when other NAC members are ectopically expressed [28].
Interestingly, when replacing the ANAC019 NAC domain with the NAC domains of NAC2/ORE1 and NTL8 plants became ABA hypersensitive, whereas introducing the SOG1 NAC domain had no effect on root elongation compared with control plants (Figure 7B). Substituting the ANAC019 C-terminal TRD with NAC2/ORE1 and NTL8 TRDs had no effect on root elongation compared with control plants. Replacing the ANAC019 TRD with the SOG1 TRD could have a lethal effect on plants or hinder T-DNA (transfer DNA) insertion events, as no transformants were recovered from several attempts to ectopically express the ANAC019–SOG1 chimaera (Figure 7B). Real-time RT–PCR analysis was used to analyse whether the observed phenotypes correlated with the chimaeric transcript dosage. All transcripts were expressed in the same range, although transcripts of ANAC019-(1–168)–NTL8-(158–335) and NAC2/ORE1-(1–174)–ANAC019-(169–317) showed the lowest abundances (Supplementary Figure S1). In conclusion, these results imply that the C-terminal region of ANAC019 encodes a major level of functional specificity alone and when fused to both distantly and closely related NAC domains of ABA-inducible origin.
In this manuscript the modular structure of plant-specific NAC transcription factors was parsed to improve the understanding of their structure–function relationship. Most NAC proteins have an N-terminal NAC DBD and a C-terminal TRD. Such modularity is a paradigm of TF architecture, and members of individual TF families often target similar core cis-elements [1]. In accordance with this, DNA-binding studies of NAC proteins from distant clades have shown that NAC TFs bind a palindromic consensus sequence containing a core CGT[GA] sequence [15,16,36,37], supporting the existence of conserved TF family DNA-target sites. However, CASTing using the barley NAC protein IDEF2 and the Arabidopsis calmodulin-binding NAC CBNAC (a group I protein) selected unrelated NAC-BSs with a GCTT core sequence [46,47]. In the present study the structural divergence of the NAC proteins was used to examine their DNA-binding properties using a consensus sequence selected by two NAC proteins, ANAC019 and NAC2/ORE1, which belong to different NAC groups (Figure 1). Apart from the ANAC019 and NAC2/ORE1 NAC domains, the NAC domain derived from ATAF1 and VND7 bound palNAC-BS with comparable affinity, whereas the rest of the NAC domains bound this oligonucleotide probe with lower affinity or, in the case of ANAC003, with no detectable affinity. This difference in affinity cannot be explained by the quality of the recombinant proteins which were all soluble, homogenous and of high purity. Therefore the most probable explanation is that NAC TFs from different groups recognize different DNA-target sites.
The findings in the present paper raise other intriguing themes associated with transcriptional regulators. The phylogenetic analysis of the NAC domains shows the major subgroups correlate with conserved motifs in the C-terminal TRDs of NAC proteins (Figure 2B and Supplementary Table 2). Although divergent with respect to sequences, these motifs have common features including a dominance of polar residues and a few highly conserved hydrophobic residues, characteristic of TRDs [2]. The analyses also suggested that C-terminal NAC regions have a high degree of ID. On the basis of these properties, the NAC proteins are well-suited as model proteins for systematic analysis of structural ID, a research topic of great current interest. Most NAC TRDs contain sequence motifs that may be recognized both by specific and general proteins of the transcriptional apparatus, and, in cases such as motif l (Figure 4B), where they interrupt the large disordered TRD, they could serve as MoRFs for partner-induced protein folding [41]. A few of these motifs, e.g. motif W and motif WQ, have already been shown to be essential for TRD activity [19]. Further characterization of these motifs may depend on the identification of interaction partners; screening using motif-based consensus sequences could represent a useful strategy for identifying such interactors.
The phylogenetic analysis also revealed tissue-specific NAC gene expression clusters correlating with phylogenetic clades (Figure 1). Such observations have been reported for members of other large gene families [48] and suggest that the NAC gene family has expanded throughout evolution by gene duplication creating paralogous genes with a high degree of sequence similarity and functional redundancy. However, the less prominent correlation between phylogenetic clusters and stress expression patterns could indicate that functional redundancy of regulators from the same tissue is diluted during the fine-tuned environmental stress perceptions needed for an adequate response of the given plant tissue. Future systems-orientated temporal studies of selected tissues in response to environmental stresses are needed for an improved understanding of overlapping NAC gene expression patterns and potential functional redundancy.
Another interesting observation from the characterization of NAC TF modularity was the ability of ANAC019 DBD and TRD to mimic ABA hypersensitivity associated with ectopic expression of full-length ANAC019 in a wild-type background (Figures 6A and 6B). This suggested that powerful effects may result from engineering of NAC TFs. The ability of the ANAC019 DBD to positively regulate ABA signalling may be explained by its ability to dimerize with endogenous NAC domains and thereby enhance promoter binding [15] and subsequent activation of ABA-signalling genes. In contrast, the ability of a NAC TRD to function without its natural molecular context has not been reported previously. The functional specificity may be explained by the ability of isolated regions to functionally interact with other proteins of the transcriptional complex. That specific interactions are important for ANAC019 TRD function is supported by the inability of the TRD from NAC2/ORE1, NTL8 and SOG1 to functionally replace ANAC019 TRD in the chimaeric proteins (Figure 7B). Although knowledge of how the isolated ANAC019 TRD exerts its function in vivo remains elusive, it is intriguing to acknowledge research performed more than two decades ago on the GAL4 transcription factor [3]. Herein, an N-terminally truncated version of GAL4, disabled in DNA-binding, was able to induce the expression of a reporter gene and even localize to the nucleus when overexpressed in a GAL4 wild-type background [3]. All together, Johnston et al. [3] speculated that the truncated GAL4 protein was transported into the nucleus in association with the wild-type GAL4. This scenario is somewhat analogous to our findings on the expression perturbations of ABA- and stress-signalling reporter genes induced when full-length and truncated versions of ANAC019 are ectopically expressed (Figure 6C). Additionally, the hypothesis on co-transportation should be addressed by analysing the subcellular localization of ANAC019-(169–317) and reporter gene expression in a 35S:ANAC019-(169–317)-expressing anac019 mutant background.
Although truncated versions of ANAC019 excluding either the DBD or the TRD of ANAC019 retain ABA hypersensitivity when ectopically expressed in a wild-type background, the potential of the ANAC019 TRD is of particular interest. In addition to its autonomous effect when ectopically expressed, the ANAC019 TRD encodes a major level of functionality during ABA signalling even when fused to NAC domains from NAC2/ORE1 and NTL8 which are members of distantly related groups. The chimaera, NAC2/ORE1-(1–174)–ANAC019-(169–317) and NTL8-(1–157)–ANAC019-(169–317), also mimic the ANAC019-mediated phenotype of ABA hypersensitivity. In contrast, replacing ANAC019-(1–168) with SOG1-(1–215) did not confer ABA hypersensitivity when fused to the ANAC019 TRD. Although seemingly contrasting with the results showing that ability of CUC proteins to enhance adventitious shoot formation is dependent on the NAC domain [19] we propose that the functionality of the NAC domain of ANAC019 during ABA signalling is exchangeable with NAC2/ORE1 and NTL8 NAC domains due to the conservation of key residues. Hence, several studies have demonstrated that NAC proteins form both homo- and hetero-dimers [5,13,39], and the structure of the NAC domain fold contains both dimerization and DNA-binding interfaces [13,15]. In the NAC domain salt-bridge, Arg-19, Glu-26 and conserved hydrophobic residues are important for dimerization [15]. Arg-19 of ANAC019 is conserved in 81% of the NAC proteins, including NAC2/ORE1 and NTL8. In SOG1, and the additional subgroup IX-1 members, Arg-19 is replaced by a lysine residue and the sequence of the dimerization region shows a relatively low degree of conservation in these proteins (Figures 2A and 2D). This supports the hypothesis that residues in this region play an important role in the 'exchangeability' of the ANAC019 NAC domain with the NAC domain of NTL8 and NAC2/ORE1 during ABA signalling. This is further substantiated by our previous demonstration of in vitro ANAC019–NAC2/ORE1 heterodimerization [15]. Future studies using site-directed mutagenesis of SOG1, NTL8 and NAC2/ORE1 are needed to fully elucidate the roles of these residues.
The DNA-binding motif of the NAC domain has not been finally identified and no atomic-level structure information is available for a NAC–DNA complex. However, studies of mutant ANAC019 NAC domains pinpoint the loop/β-strand region containing Lys-79, Arg-85 and Arg-88 of ANAC019 as the DNA-binding, or part of the DNA-binding, motif (Figures 2A and 2D; [15]). Whereas Arg-88 is conserved in all NAC proteins, Arg-85 is replaced by a glutamine residue in NTL8 and ANAC003, and Lys-79 is replaced by an alanine residue in SOG1 and VOZ2. These changes may explain the decreased palNAC-BS affinity of these proteins. As Lys-79, Arg-85 and Arg-88 are conserved in NTL6 and VND3, which also bound palNAC-BS with lower affinity, additional regions or residues, such as Arg-78 or Arg-76, must be of importance to DNA binding. It is possible that the ability to dimerize with endogenous ANAC019 could overrule suboptimal DNA binding by, for example, the NTL8 NAC domain in enabling ANAC019-mediated ABA sensing. However, in the case of the SOG1 NAC domain, which binds palNAC-BS with relatively low affinity, dimerization may not be sufficient to compensate for weak target-DNA binding. Although ANAC019 mutants deficient in dimerization cannot bind cognate DNA-binding sites in vitro, it is not known whether two NAC-BSs are needed for in vivo DNA-binding by NAC TFs [15]. Hence, although the C-terminal TRD of ANAC019 is needed for conferring ABA hypersensitivity in NAC proteins with both a DBD and a TRD, residues in the NAC domain could modulate this activity through inter-domain communication for identification of interaction partners and target promoters from which the ANAC019 TRD exerts its transactivation activity. All together, our results have laid out future directions for the engineering of NAC TFs for an improved understanding and use of NAC networks and for investigating their role in plant stress perception.
abscisic acid
Arabidopsis Biological Resource Center
- CaMV
cauliflower mosaic virus
- CASTing
cyclic amplification and selection of targets
- Col-0
Columbia ecotype 0
cold-responsive 47
cup-shaped cotyledon
DNA-binding domain
electrophoretic mobility-shift assay
early-responsive-to-dehydration 11
ferritin precursor 1
glutathione transferase
intrinsic disorder
motif alignment and search tool
multiple expectation maximization for motif elicitation
- MoRF
molecular recognition feature
NAM (no apical meristem), ATAF, CUC
NAC-binding site
- palNAC-BS
palindromic NAC-BS
position-specific iterated
- Rap1p
repressor activator protein 1
- RD29b
responsive-to-desiccation 29b
reverse transcription
suppressor of gamma response 1
synthetic defined
The Arabidopsis Information Resource
tiling array express
transcription factor
transcription regulatory domain
vascular-related NAC domain
vascular plant one-zinc finger protein
The planning of experiments and writing of the manuscript were performed by Michael Jensen, Trine Kjaersgaard and Karen Skriver. Michael Jensen, Trine Kjaersgaard, Pernille Galberg, Charlotte O'Shea and Michael Nielsen contributed to experimental characterization of NAC structure–function. Klaus Petersen and Michael Jensen performed work relating to gene expression analysis.
We thank Dr Lars Ellgaard for critical revision of manuscript prior to submission.
This work was supported by the Danish Agency for Science Technology and Innovation (DFF, Technology and Production) [grant numbers 274-07-0173 (to K.S.) and 274-07-0385 (to M.K.J.)].


text:
Deni Goure - Chatham-Kent Cyclones
- Details
- Created: Thursday, 04 October 2018 18:50
Deni Goure
Chatham-Kent Cyclones #7
Position: RW/C
Shoots: R
July 15, 2003
5'10", 160 lbs
Grande Pointe, ON, CAN
An electric offensive talent, Goure dazzles in possession and is one of the most lethal goal-scorers in the 2003 age group. A dangerous threat every time on the ice, Goure is a player who maximizes his elite skill-set to the fullest, whether through his nifty edges and quick feet, quick hands, instinctive vision, or pro-level shot.
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subset: commoncrawl


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Book - Gardening in Miniature: Create Your Own Tiny Living World
Book - Gardening in Miniature: Create Your Own Tiny Living World
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Our Mission
To provide affordable and appropriate homes primarily for Aboriginal people on Vancouver Island, and to enhance community partnerships.
About M'AKOLA HOUSING SOCIETY
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To provide homes* primarily for Aboriginal people, where they can have pride and feel connected to traditional values and a community.
* "Homes" includes a range of residential programs and services, including those that provide appropriate care and support


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General Info
Welcome to the Mikhail In-Depth Guide. In this guide, you'll learn all you need to learn about Indines' Battle Hand of Divinity! Note that this guide is NOT a substitute for experience. It's always better to go out there and play some games!
About the Author
This guide was written by me, Marco AKA mnmkami AKA TheMechanicritic! I have a ton of BattleCON experience under my belt. I also consult some of the known players to confirm the information presented in these guides!
Quick Reference
The Hand of Divinity does well against fighters who cannot keep-up with his amazing trading and dodging power. This means characters with limited damage, or limited time, such as Karin and Clinhyde respectively, have trouble dealing with him.
Mikhail's problematic match-ups come from characters who are easily able to control the pace of the game. These control characters make sure that, when Mikhail decides to use his Seal Tokens, he wastes them on nothing. Characters like Marmelee and Sagas are great examples of this.
Mikhail is a mega heavy who has absolutely overpowered styles. However, to even use them, he must pay for it with a Seal, which can only be regained by taking damage.
Simple Tips
Trade often. Mikhail is powerful, no doubt, but he can only afford to step on the gas for so long. To ensure a stable power level, he must take damage to regain Seal Tokens. As such, do try to trade with your opponent as much as possible.
Scroll up! Scroll, Mikhail's unique base, is the only way to regain Seal Tokens without taking damage. As a result, the base has below average stats, which are often mitigated by his styles' amazing effects. Scroll also allows Mikhail to regain Seal Tokens even if the opponent dodges, making it a good counter to one of the biggest forms of counter-play against him.
- You don't always need a style! Just because they're powerful, it doesn't mean that you need to always apply your style to your attack. Sometimes, it is actually better to simply save the Seal for a future beat. The most obvious example of this is when using Dodge, but there are many more.
- Massive trading capability
- Uncanny ability to dodge or make the opponent miss
- Is awfully well-balanced for being a Heavy
- Styles can have awkward use cases
- Can't win by too much in a single beat or he won't regain his Seals
- Anteing a Seal when the opponent Dodges can be a huge setback
Role on the Team
Mikhail serves a really simple role on the team in that he does not have any particularly egregious match-ups. His bad match-ups are hard to deal with, but not down-right impossible. With correct reads and play, Mikhail can perform against nearly anyone. Such is the strength of his powerful styles. As such, Mikhail fulfills more of a "fill" role on the team, providing a consistent character that can somewhat be useful in a variety of situations.
Card Analysis
Unique Ability
Mikhail's Unique Ability is Sealed Power. He starts the game with 3 Seal Tokens and must ante one in order to apply his style to his attack. Otherwise, he's essentially playing a naked base. In order to regain them, Mikhail must either take damage or play his Scroll base.
More than anything, this Unique Ability is an obvious drawback meant to mitigate just how powerful Mikhail's styles are. However, this Unique Ability has one edge use.
By not applying his style to his attack, Mikhail, essentially, gains on-demand access to a Range +0, Power +0, Priority +0 style with no text. This can be useful if the styles in your hand have Priorities lower than 0 and you need to be fast, for example. Looking for such opportunities can prove to be a huge boon.
Sacred might seem like such a simple card, but there's oh so much more to it. You see, many similar cards often come with a few drawbacks. Attacks that allow a character to instantly dodge attacks at range X or greater usually have poor Priority to make you rely on the dodge more. Also, that X is usually 4. Finally, they sometimes come with a specific condition that triggers the dodge effect.
With all this in mind, it's not hard to see why Sacred is powerful: it doesn't have ANY of these drawbacks! Sacred just works at a lot of ranges due to its effect and range, making Mmikhail absurdly hard to fight against at range 3 or greater. Use this style to pressure your opponents into advancing into you. That, however, may not prove to be such a great idea.
Hallowed is precisely the reason why doing that might be a huge mistake. Not only will the opponent take over 6 points of damage, they'll likely just be pushed back to the edge of the board, setting-up for another Sacred beat.
Hallowed is Mikhail's strongest melee option. It deals an absurd amount of damage at a respectable speed. On top of that, it has nearly magical Range, giving his attacks range +0~2 when not dealing with Bursts.
However, do not be fooled, this style STILL works, even at long range, giving Mikhail a LOT of options to dish out divine punishment.
Immutable is probably one of Mikhail's weaker styles. You heard that right. One of Mikhail's weakest cards is essentially Cadenza's Clockwork with less Power and more Range.
This style is quite powerful as it gives Mikhail another absurd trading tool with an effective Power +5. The Soak also proves useful in the late game, when the slow-going Mikhail can have trouble against speedy executioners.
What makes this style less powerful than the rest is how taxing it is on Mikhail's Seal management. Unlike his other styles, Immutable requires 2 Seal Tokens to function optimally as you have to spend one to apply the style, then spend another to get that Soak. Finally, Soak, strangely, is counter-productive to Mikhail's strategy as he must take damage in order to regain his Seals.
Speaking of cards that are awfully similar to Cadenza's, Transcendent essentially turns anything Mikhail plays into Cadenza's Press. What makes this immensely better than Press is the fact that it can be paired with nearly ANY base. This is due to two big factors: Range and Stun Guard.
Transcendent has magical Range +0~2, meaning that it is effective at nearly any range while also enabling low-range bases like Grasp and Strike. It also has built-in Stun Guard 5 (way beyond the magical value of Stun Guard 3). This makes Transcendent quit versatile and powerful as it nearly always allows Mikhails to trade positively against any attack.
This attack does have one glaring weakness: it might be too fast. Given that Mikhail must take damage in order to gain the bonus Power, he must, therefore, be the slower character. As such, be careful when pairing this with Grasp or Drive.
Apocalyptic has a very fitting name, seeing as its stats are absurdly strong. If it feels awfully familiar, do not fret! It is, in reality, very similar to Immutable! The key different between them is Apocalyptic's fast movement and its Range.
Apocalyptic gives Mikhail the ability to slightly dodge attacks at range 2 while also setting-up and dealing a considerable amount of damage to the opponent. Did I mention that it ALSO has Soak and Stun Immunity on top? This attack will barely lose any trades. One simply just has to watch out for opponents with advancing effects that can easily put them in the attack's minimum range.
However, to offset this style's incredible power, it prevents Mikhail from regaining Seals on the beat he uses it. Though, it is still effectively cheaper to use than Immutable!
Mikhail's Unique Base, Scroll, is an odd card because, unlike many of his cards, it has poor stats. With only 2 Power and Priority, Scroll will not be trading-up against many things.
Its biggest use comes in its ability to guarantee a Token regain, regardless of what happens. This can be very good in ensuring that Mikhail is able to continue his assault over the next few beats.
Specifically, Scroll is a very good counter-play to opponents who are a little too Dodge-happy.
Force Special Action (Switch)
Mikhail's always had access to a "blank" style thanks to his Unique Ability, meaning that the addition of Switch does not actually benefit him much.
If anything, the biggest thing is that Mikhail is now able to hold 4 styles in his hand. Seeing as his styles are absurdly good and exert a sizeable amount of pressure, having 4 can quickly force opponents to crumble.
The 4th Seal is Mikhail's "no more resources" Finisher as it only hits when he has no Seals left. Seeing as a Mikhail without any Seals is completely powerless, this can serve as a great trump card to finish-off a cocky opponent. Its Range, Power, and Priority are all above average, making it a decent late-game execute as long as the opponent's Burst is down. Just remember that, unlike Mikhail's other attacks, this one is dreadfully "honest", meaning that it has little to no trickery. The only trick it seems to have is that Mikhail can sneakily play this Finisher even if he has one Seal. He simply has to ante it during the ante phase.
Mikhail is most comfortable in mid to long range, making Magnus Malleus the go-to when keeping your distance. As a plus, it also scales in power depending on how many Seals Mikhail has left. This means that it can possibly deal over 10 damage! Its above average speed and intense power allow Mikhail to threaten people with a swift death so long as he has the Seals to back it up. One thing to remember is that, unlike Mikhail's attack pairs, he doesn't need to ante a Seal to make this attack work. Instead, he can choose to spend Seals once the attack has already hit, making this safer in terms of Seal expenditure.
Content & Editing - Marco De Santos


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When it comes to the offseason in sports, no one does it better than the N.B.A. Fans spend countless hours refreshing their Twitter feeds in hopes of seeing breaking news regarding their favorite teams. We decided to get in on the craze by announcing McDonald's very own SuperTeam being formed on the new McPick menu.
Concept / Art Direction: Carolina Perez-Siam
Concept / Copywriting: Eddie Seiglie
ECD: Jorge Murillo & Monse Valera
DDB Alma / June 2017


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We all have bucket list experiences or achievements set in the horizon and no matter how simple or complex your goal is, there is no better feeling than living and enjoying that experience. The White Marlin Open was a bucket list item and in 2018, the stars aligned and what an awesome tournament it was.
The Richest Billfish in the World
Step one foot into any bar or restaurant in Ocean City, Maryland the first full week of August and the talk is centered on the White Marlin Open. Every square inch of dock at Sunset Marina has a beautiful sportfish yacht tied up with crews prepping bait and money flowing like water at Teasers. The big boys are in town to win it all. How much money? The total purse for 2018 was $5.45 million.
Teams strategically enter into the various divisions, daily categories, points, and multipliers to increase the odds for their targeted species or focus angling. Early base entry was $1,050 but with little in the pot, the potential for big money winnings is minimal. To go all in would cost you $35,000! The average entry based on the purse size over 383 registered boats was $14,230.
White Marlin Hunters
It was eye opening to see the 80+ft sportfishing vessels docked at Sunset. They were in town to hunt and win, no other agenda. Every mate and crewman had a role and even on lay days (you can only fish 3 out of 5 days), they were working on bait prep and meticulously washing down the boat and gear.
Canyon Hunter Sportfishing
I had the opportunity to climb aboard the Canyon Hunter, a 41ft Luhrs Express, captained by Travis Scott out of Indian River Inlet. Two mates Adam and Steve handled the bulk of the prep and legwork as I soaked in the experience (didn't want to get in the way). The fishing days started with a 2:00am wake up and at the dock box by 3:15am. By 4am we were waiting inside the sea buoy of the Indian River Inlet and at 4:01am, the twin Cummins diesels throttled up and we were heading East! I was impressed with the energy and personal drive of Travis and the mates. This wasn't another charter for them, this was make or break – they wanted to get to the scales come hell or highwater. Intensity and passion were at 110% and it was contagious.
Day 1 – Monday Aug 6, 2018
Exact location unknown, but it was 80+ miles South East out from the IRI sea buoy. The first catch was a small mahi we boated for tacos. Later we released two white marlins, both of which were under the tournament length requirements. The first white put on a show for us, dancing out of the water, pulling line and charging the boat. We chalked up the first day as a success.
Day 2 – Wednesday Aug 8, 2018
After taking a lay day Tuesday for some minor engine servicing, we were back at it. Although not as far out as Monday, it was still was an adventure with some sporty sea conditions. Yeah, I popped two Dramamine… she was rocking and rolling. But after a few hours, the seas calmed down and apparently so did the bite. Nothing all day until approximately 2:50pm when the Penn 80 started to scream. The boat went from dead silence to a roar in a fraction of a second… go time boys! Angler C.J. had the honors to reel in a 55lb Wahoo of which we instantly knew to point the boat to the Ocean City, Maryland inlet. With the Wahoo boated, it was throttles down and good times aboard the Canyon Hunter.
Not only was it amazing to be in the tournament, but we were really going to the scales! The energy couldn't be any higher as we crest the final dock at Harbor Island and slid into the scales. All we could see was a mob of people excited for what was on deck. The scale stopped at 55lbs, currently taking first place in Wahoo and daily meat, and the crowd was going wild. Bucket list complete!
We were later bumped to 2nd on the Wahoo but Travis played the cards right and we held onto sizeable winnings. Needless to say, we were not going to be able to physically wake up in time for a Thursday run.
Day 3 – Friday Aug 10, 2018
Although the passion and drive was there, the bite was not on our third and final day. One early bite was all that was had for the day. Although humbling, we still accomplished what many boats did not and we had a heck of a good time doing it.
When it was all said and done, we finished 3rd in the Wahoo division and took the Wednesday daily meat winning us a total of $21,471 and an evening at the awards ceremony. It was an incredible experience to be in the White Marlin Open and recommend anyone who loves fishing to do it once in their lifetime. The prize winning and trip to the scales was just icing on the cake! Will I do it again, ABOSULTELY! It was also great to see families involved not just directly with a boat or angler, but even as a spectator. Children were all over the scale docks waiting for the next fish to be hoisted out of the fish box. It really is something to see even if you just stop by the scales one evening.


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Friday Inspiration: Our Top Pinned Images
This week has been busy and productive for our team! We got the flu shot on Monday - gross! We delivered a fall wedding design to a client on Wednesday. Today, we're meeting with some friendly planners. And, later this afternoon, we're touring Scarritt-Bennett for the first time!
This weekend will be slow and easy as we try to recover from a cold virus and catch up on the dreaded laundry. To pull us out of the funk and into spring state of mind, we're pinning so many colorful ideas for 2018!
A napkin fold we'll have to try with that perfect wax seal.
Because last week's garden inspiration wasn't enough . . .
Taking notes for a future party in my backyard.
Delicate details.
Taper candles are back and here to stay.
Make sure to follow us on Pinterest for daily inspiration!


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A Geometer's Sketchpad of a right triangle that illustrates Pythagorean's Theorem. Users can drag any vertex of the triangle and watch the calcualtions for the square of each leg, the sum of those squares, and the square of the hypotenuse change dynamically.
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Hsu adds to score of musical feats
Violinist Rachel Hsu will play at Carnegie Hall over winter break
December 22, 2017
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The old joke goes like this: a man is walking around New York City. He asks a policeman how to get to Carnegie Hall. "Practice, practice, practice," the policeman says.
That is exactly what junior Rachel Hsu has done.
Her recent success is due to her lifelong love of music. When she was three years old, she hoped to be like her older brothers, Sam and Jeremiah, who play violin and piano.
By first grade, she realized that her commitment to music was exceptional. "Even when I was in elementary school, I realized that I played way more than anyone else."
Her passion continued until sixth grade, when Hsu experienced a plateau in her motivation. "I had an 'I hate music' phase," said Hsu. Music had always restricted her social life and hindered her from trying other extracurriculars.
It was around this age that Hsu started playing with the Chicago Youth Symphony Orchestra (CYSO). The groups she was in, the Encore Chamber Orchestra and Symphony Orchestra, practiced from 2 to 8 PM on Sundays and throughout the week.
Hsu eventually quit piano. "I didn't have competitions to aim for, so there were no end goals," she explained. "Competitions, concerts, and auditions are what drive me."
It wasn't until the summer after freshman year that Hsu rediscovered her passion for music.
Her violin teacher invited her to perform with the Filarmónica Joven De Colombia, the national youth orchestra of Colombia. The orchestra consisted of serious musicians from 17 to 25 years old. Most were already in school for music or were playing professionally.
Hsu thrived in such an intense environment. "I had never been in an orchestra surrounded by people who want to do music for their lives," she said. "They chose music."
This orchestra also felt much less competitive feeling than the other ones Hsu had played for. "It was a huge community. Everyone was friends."
Hsu was recently accepted into the New York String Orchestra Seminar. The group consists of musicians who are 16 to 23 years old. Most of them are pursuing degrees in music.
On Dec. 24 and 28, they will perform at Carnegie Hall.
Hsu has been concertmaster the past two years at the Illinois Musical Educational Conference.
She was second violin principal in the New Trier Symphony Orchestra freshman year, one of the concertmasters sophomore year, and is currently co-concertmaster.
"Her skill set is really fantastic in terms of her ability to play the instrument technically," said Orchestra teacher Peter Rosheger.
Senior and co-concertmaster Ria Honda explained that they share many responsibilities. "She always gives me her honest opinion and helps me make important decisions. I think we make a great team," said Honda.
Before big competitions or recordings, Hsu practices about two hours per day for at least a week.
When she is not in school, she is touring the world with various orchestras.
Last summer, Hsu toured Central Europe with CYSO. The group of about 100 did concerts in Belgrade, Budapest, and Prague.
Soon after, she attended a musical festival in Simorre, France started by her teacher Desiree Ruhstrat. Hsu performed in many different concerts as a soloist and in chamber groups.
Rosheger added that Hsu not only has an excellent skill set, but also uses her experience and mastery to "provide insight and confidence to the rest of the orchestra."


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Outreach Sewing Day will be held on Monday, September 28 at the Gmeiner Art & Cultural Center from 10:00 until 2:00. As always, all guild members are welcome and encouraged to attend. Bring your own charity project to work on, sewing supplies, and a bag lunch.
Contact Dori S. for more information. (check your roster for contact information or comment on this blog post).


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Happy birthday, baby! This twist on our original classic birthday cake features traditional yellow butter cake slathered in layers of rich chocolate buttercream, all dolled up with cheery rainbow jimmies. No better way to celebrate!
Chocolate Birthday Cake
$45.00 – $85.00
All our cakes consist of 3 layers of cake and 2 layers of icing. Please note that we typically require 2-3 weeks notice for cake orders.
Additional information
6", 8", 9", 10"
Store cakes in the refrigerator until 3 to 4 hours prior to your party. We recommend enjoying your cake at room temperature.


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Howdy y'all (ooo how American of me lol!) I hope you are doing fabby in this fresh weather? I think today was the coldest for me. Mum and I popped over too Dedham, well really too the farm shop that is up the road.
We ordered a capon chicken, which I have never had, it was recommended to me that they are much nicer than your regular clucker.. So that's all ordered and ready to be picked up for Christmas. I used to buy turkey but seen as its really just Mum, Sonny and me who eat it, lots goes to waste. So I get an extra large chicken which is my preference anyway. Can I just say the stuff in the farm shop is so scrummy but also ridiculously priced. We literally go there for a treat a few times a year.
Anyhoooo guess what I did… No! OK then let me tell you. While I was farting about I happened to stumble across this site.. I AM IN LOVE, the wonderful talent who draws these little scrummies is Linnette and her little site Paper Glitter. There is so much colour on this site my eyes were just popping. Check her out and let me know what you think.. I downloaded some Christmas check lists which are sooooo handy, Thank you Linnette x
Anyhooo Happy Saturday and have a wonderful Sunday x


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A good decision is based on knowledge, not numbers.
In the previous post we discussed some important foundational ideas when talking about cholesterol. Today we'll start going into all this just a bit deeper, starting with what all those numbers mean and why I say that your total cholesterol number is meaningless for you, personally.
What sadly happens to way too many people is this:
First, they get a cholesterol test.
Second, they get an ominous call that their test results are in. They are given their total cholesterol number–something over 200, which it should be unless they're sick–with a warning that they ought to talk to their doctor about this immediately. There are some other numbers thrown around, perhaps. Maybe. Some talk of HDL, or LDL, or triglycerides. But a lot of times those are kind of ignored, and sometimes you may even have to specifically ask them to tell you the breakdown, so focused are they on that scary total number.
Perhaps the most disturbing thing in all is how many in the health care profession have taken to looking at these surrogate markers–because that's all cholesterol testing does–and become obsessed with making the marker levels change instead of dealing with the underlying health issues involved. This is as if your white blood cell count were abnormally high and instead of your doctor looking to see if you might have a serious disease or infection, he instead prescribed you a drug that lowered white blood cell count and told you to come back in a month.
So what is cholesterol? In terms of heart health, here's what you need to know:
In the beginning, you get inflammation in your arteries. This could be for a vast variety of reasons, from lack of sleep, to smoking, to stress, to eating inflammatory foods–particularly grains and sugars–to just generally having poor glycemic control, too much iron in your blood, or too much Omega-6 and/or not enough Omega-3 fats.
Your body responds to these inflammations and damaged arteries the way God designed it to: lipoproteins start carrying cholesterol around. LDL (or low density lipoprotein) carries the cholesterol to the damaged area to repair it. LDL does not damage arteries in any way, nor does it trigger an inflammatory state. If you didn't have plenty of LDL available to do this chore, you wouldn't die of an arterial plaque rupture at 50–instead you'd get to die from arterial failure in your 20s. Maybe earlier.
HDL (high density lipoprotein) then carries the used cholesterol away. Obviously this is important. You can't have the LDL just laying around there building up all the time. Having enough HDL is far more important to your heart health than your LDL or total cholesterol levels. But neither LDL and HDL are "bad" or "good." You need both.
You should also know that LDL comes in different sizes. The smaller LDL (sometimes known as VLDL, or very low density lipoproteins) is easily oxidized, so that's something you don't want too much of. That's because while there is no link between LDL levels and heart disease, there is a link between oxidized LDL and heart disease.
Oxidize is a fancy name for "rust." It's what happens when an oxygen atom combines with anything, and whether it's the inside of your boat or the inside of you, oxidizing causes deleterious effects.
OOH OOH CLASS! Here's a question…if you don't want your LDL oxidizing…HOW CAN YOU STOP IT FROM HAPPENING?
Well, according to modern statin-pushing theory, the best thing to do is just make sure you don't have any LDL in your blood. Or at least as little as possible. Take a statin or don't eat any butter and your LDL will drop below 130 or so (we think) and you'll be fine! No LDL to oxidate, no oxidised LDL.
This is a brilliant solution!
I'm going to apply this solution to my moped and my bike. The reason I'm getting all that rust is because I've got all that metal in them. The answer isn't for me to keep them covered, or in any way protect the metal so it isn't exposed to oxygen. The answer is for me to just cut all that metal off! No metal to rust, no rusted metal.
Ok, obviously I'm jesting. Obviously that solution is…say it with me now..STUPID.
Instead of lowering your total LDL, why not stop doing things that cause you oxidative stress? Then your LDL won't be oxidizing at enormous rates no matter how much of it there is.
SO what can we do to lower oxidative stress? The number one, first, most important, easiest, simplest, most straightforward way to do this is to keep your blood sugar low. Chronically high blood sugar is what is causing your diabetic friend to age faster than you–he is literally rusting away at a faster rate. If you looked at that last link I posted, the one showing that it is oxidized LDL and not LDL total levels that are linked to heart disease, you'll see that those with "metabolic syndrome" were twice as likely to have high oxidized LDL levels. What is metabolic syndrome? Put simply, you can consider it the precursor to diabetes and something one in five Americans have: the inability to properly control blood sugar.
How do you lower your blood sugar? Very, very, very, very simple. (At least simple for those who haven't yet gone into full-blown diabetes). Stop eating stuff that spikes it. Don't drink sodas and fruit juices, or eat candies and sweet things, potato products and starches. Don't whirl up enormous amounts of fruit into smoothies and suck down three times as much fruit as you would normally eat in the form in which God made it for you to enjoy. Enjoy your vegetables in moderation and with plenty of healthy fat to go with them. Do not eat salads the size of your own head, especially on an empty stomach, with sugary low-fat dressings. Don't eat that ice milk garbage or that low-fat frozen yogurt nonsense. Stop downing granola bars, Special K, and low-fat milk.
Here's another reason to put down the pizza–remember I mentioned that LDL comes in different sizes? Well that VLDL kind is easily oxidized, unlike the large, fluffy LDL. It also tends to stick around your arteries longer, seemingly for two reasons: First, it's harder for HDL to carry away in the first place. Second, it's inversely proportional to HDL, meaning that whatever is causing you to produce VLDL is also lowering your HDL.
Changing the makeup of your LDL from tiny and dense to big and fluffy is much more important than lowering your total LDL level. (Or it might be more proper to say, producing large fluffy LDL instead of VLDL. I don't believe you can actually change the size pattern of an already existing lipoprotein). And whatever you do to change your LDL size pattern production also tends to change the level of your HDL, for good or bad. So what can you do?
I'm noticing a pattern here. Are you?
In case you're too busy to read that link I just gave, I'll summarize for you. Researchers took a bunch of fat, middle-aged men, took away almost all their carbs, and put them on a high fat diet. One low carb diet included added fiber, one did not. They were eating 10% carbs, 65% fat, and the rest protein. They did this for 12 weeks.
So first, these guys lost an average of 16 1/2 pounds in those 12 weeks. ("Water weight!" Can you hear them screaming it at us? "It was all water weight! That's why low carb diets give such great results!") Well, sadly for all those people whose brains have been damaged by eating low fat, these guys also reduced their measurable abdominal fat by 20%.
For our current purposes, though, what's really important is that these men saw their LDL and triglyceride levels drop, their HDL levels skyrocket, and their LDL changed in pattern from small and dense to big and fluffy and happy.
Just if you're curious, what these guys ate was:
Unlimited amounts of beef, poultry, fish, and eggs, moderate amounts of cheese, low-carbohydrate vegetables, low-carbohydrate salad dressing, and small amounts of nuts and seeds.
And, by the way, the fiber that half the guys were drinking didn't make one lick of difference in any way, shape or form to any health marker of any sort.
Let's get back on track.
I said that there's no link between your LDL levels and heart disease risk. Or between your total cholesterol level and heart disease risk. That's because there isn't.
Look here. This writer summarizes for you a bunch of different studies, all of which he cites and you can check for yourself. Just a few of them include:
1. One showing that half, HALF, of the people who get coronary heart disease have normal or low LDL levels.
2. "Among elderly Belgians, higher levels of oxidized LDL were accompanied by significantly increased risk of heart attack, regardless of total LDL levels."
3. " In Japanese patients undergoing surgery to remove plaque from their carotid arteries, blood levels of oxidized LDL were significantly higher than those measured in healthy controls. Elevated oxidized LDL was also associated with an increased susceptibility of plaque rupture. However, there was no association between oxidized LDL concentrations and total LDL levels."
4. A two-year double blind study with fish oil in people with heart disease, which found that taking fish oil every day caused arteries to stop distending and plaques to get smaller and the people themselves to experience fewer "cardiovascular events." But…their LDL cholesterol levels were higher!!!! Oh no! Give them statins, quick!
5. Two studies in which plaques were measured in the arteries. Both groups were treated with statins to lower their LDL, but one group was also given niacin to lower it even further. They succeeded–the statin/niacin group had much lower LDL. But sadly, they didn't experience any fewer or any smaller plaques than the others, leading the researchers to conclude that "with respect to LDL cholesterol lowering, 'lower is better' is not supported by changes in calcified plaque progression."
6. And finally, two groups with calcified aorta were treated, one with a statin, the other with a placebo. The ones with the placebo kept their perfectly normal, if not slightly low, 130 mg/dL level of LDL. The ones on the statin got down to 63! What an achievement! Unfortunately, it didn't make the slightest difference to their arterial health.
And here's another. From the very first study of its kind in 1957, all the way to today, no study has ever shown any benefit to mortality rates by lowering cholesterol. In fact, that first study was totally successful in getting cholesterol levels to drop, yet the ones eating the cholesterol-lowering, low fat diet got more heart disease and more of them died than the control group eating a normal, fatty diet.
And here. 100 guys from heart-disease rampant Lithuania and 100 from heart-disease free Sweden. (Relatively speaking, of course). The guys from Lithuania had lower total cholesterol and lower LDL than the Swedes. Their triglycerides were similar, and smoking rates and general health were similar. Shockingly, the Lithuanians had higher blood pressure, more LDL oxidation, and more heart attacks.
And here. Another summary of some studies, among them my favorite being the MR-FIT study, which got a huge number of men to reduce their cholesterol consumption by 42% and their saturated fat consumption by 28% and their total calorie consumption by 21% and yet saw 0% change in heart disease. Researchers referred to these results as "disappointing."
And here. A study designed to be longer-term and measure not just whether a statin and successful cholesterol lowering stopped people from having heart attacks, but whether they actually improved all-cause mortality. As we mentioned last time, no one will be happy that you didn't get a heart attack as they are burying you after you die of cancer or kill yourself in a depressive fit. The study, again, found that while the drug was super successful at lowering cholesterol, it didn't stop people from dying any faster than anyone else, and it didn't do much to stop them having heart attacks.
And here: This one is kind of sad. This one was with at-risk elderly people. Again they succeeded in getting a drug to lower their cholesterol. If you just read the abstract you'd think this was a study that proved how great it is to take this drug. But keep reading and look at the numbers.
Math'll get ya in the end, folks. That's why I don't do it…
Anyway, go down past all the stuff and look at the numbers. In the end, it made almost no difference at all whether you took the placebo or the drug, as far as your likelihood to die. 306 placebo patients died; 298 taking the drug died. And there were 22 more patients taking the placebo than the drug, anyway. Since we're talking about elderly people (70+ years old), we really have to wonder if the side effects of this powerful drug were really worth the infinitesimally lower risk of death.
Don't worry, there's more coming…
Plato says he's hungry
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