An unconventional proanthocyanidin pathway in maize.

Proanthocyanidins (PAs), flavonoid polymers involved in plant defense, are also beneficial to human health and ruminant nutrition. To date, there is little evidence for accumulation of PAs in maize (Zea mays), although maize makes anthocyanins and possesses the key enzyme of the PA pathway, anthocyanidin reductase (ANR). Here, we explore whether there is a functional PA biosynthesis pathway in maize using a combination of analytical chemistry and genetic approaches. The endogenous PA biosynthetic machinery in maize preferentially produces the unusual PA precursor (+)-epicatechin, as well as 4ß-(S-cysteinyl)-catechin, as potential PA starter and extension units. Uncommon procyanidin dimers with (+)-epicatechin as starter unit are also found. Expression of soybean (Glycine max) anthocyanidin reductase 1 (ANR1) in maize seeds increases the levels of 4ß-(S-cysteinyl)-epicatechin and procyanidin dimers mainly using (-)-epicatechin as starter units. Introducing a Sorghum bicolor transcription factor (SbTT2) specifically regulating PA biosynthesis into a maize inbred deficient in anthocyanin biosynthesis activates both anthocyanin and PA biosynthesis pathways, suggesting conservation of the PA regulatory machinery across species. Our data support the divergence of PA biosynthesis across plant species and offer perspectives for future agricultrural applications in maize.

Leaf layer-based transcriptome profiling for discovery of epidermal-selective promoters in Medicago truncatula.

MAIN CONCLUSION: Transcriptomics of manually dissected leaf layers from Medicago truncatula identifies genes with preferential expression in upper and/or lower epidermis. The promoters of these genes confer epidermal-specific expression of transgenes. Improving the quality and quantity of proanthocyanidins (PAs) in forage legumes has potential to improve the nitrogen nutrition of ruminant animals and protect them from the risk of pasture bloat, as well as parasites. However, ectopic constitutive accumulation of PAs in plants by genetic engineering can significantly inhibit growth. We selected the leaf epidermis as a candidate tissue for targeted engineering of PAs or other pathways. To identify gene promoters selectively expressed in epidermal tissues, we performed comparative transcriptomic analyses in the model legume Medicago truncatula, using five tissue samples representing upper epidermis, lower epidermis, whole leaf without upper epidermis, whole leaf without lower epidermis, and whole leaf. We identified 52 transcripts preferentially expressed in upper epidermis, most of which encode genes involved in flavonoid biosynthesis, and 53 transcripts from lower epidermis, with the most enriched category being anatomical structure formation. Promoters of the preferentially expressed genes were cloned from the M. truncatula genome and shown to direct tissue-selective promoter activities in transient assays. Expression of the PA pathway transcription factor TaMYB14 under control of several of the promoters in transgenic alfalfa resulted in only modest MYB14 transcript accumulation and low levels of PA production. Activity of a subset of promoters was confirmed by transcript analysis in field-grown alfalfa plants throughout the growing season, and revealed variable but consistent expression, which was generally highest 3-4 weeks after cutting. We conclude that, although the selected promoters show acceptable tissue-specificity, they may not drive high enough transcription factor expression to activate the PA pathway.

MtGSTF7, a TT19-like GST gene, is essential for accumulation of anthocyanins, but not proanthocyanins in Medicago truncatula.

Anthocyanins and proanthocyanins (PAs) are two end products of the flavonoid biosynthesis pathway. They are believed to be synthesized in the endoplasmic reticulum and then sequestered into the vacuole. In Arabidopsis thaliana, TRANSPARENT TESTA 19 (TT19) is necessary for both anthocyanin and PA accumulation. Here, we found that MtGSTF7, a homolog of AtTT19, is essential for anthocyanin accumulation but not required for PA accumulation in Medicago truncatula. MtGSTF7 was induced by the anthocyanin regulator LEGUME ANTHOCYANIN PRODUCTION 1 (LAP1), and its tissue expression pattern correlated with anthocyanin deposition in M. truncatula. Tnt1-insertional mutants of MtGSTF7 lost anthocyanin accumulation in vegetative organs, and introducing a genomic fragment of MtGSTF7 could complement the mutant phenotypes. Additionally, the accumulation of anthocyanins induced by LAP1 was significantly reduced in mtgstf7 mutants. Yeast-one-hybridization and dual-luciferase reporter assays revealed that LAP1 could bind to the MtGSTF7 promoter to activate its expression. Ectopic expression of MtGSTF7 in tt19 mutants could rescue their anthocyanin deficiency, but not their PA defect. Furthermore, PA accumulation was not affected in the mtgstf7 mutants. Taken together, our results show that the mechanism of anthocyanin and PA accumulation in M. truncatula is different from that in A. thaliana, and provide a new target gene for engineering anthocyanins in plants.

Dual activity of anthocyanidin reductase supports the dominant plant proanthocyanidin extension unit pathway.

Proanthocyanidins (PAs) are plant natural products important for agriculture and human health. They are polymers of flavan-3-ol subunits, commonly (-)-epicatechin and/or (+)-catechin, but the source of the in planta extension unit that comprises the bulk of the polymer remains unclear, as does how PA composition is determined in different plant species. Anthocyanidin reductase (ANR) can generate 2,3-cis-epicatechin as a PA starter unit from cyanidin, which itself arises from 2,3-trans-leucocyanidin, but ANR proteins from different species produce mixtures of flavan-3-ols with different stereochemistries in vitro. Genetic and biochemical analyses here show that ANR has dual activity and is involved not only in the production of (-)-epicatechin starter units but also in the formation of 2,3-cis-leucocyanidin to serve as (-)-epicatechin extension units. Differences in the product specificities of ANRs account for the presence/absence of PA polymerization and the compositions of PAs across plant species.

Dissecting the transcriptional regulation of proanthocyanidin and anthocyanin biosynthesis in soybean (Glycine max).

Proanthocyanidins (PAs), also known as condensed tannins, are plant natural products that are beneficial for human and livestock health. As one of the largest grown crops in the world, soybean (Glycine max) is widely used as human food and animal feed. Many cultivated soybeans with yellow seed coats lack PAs or anthocyanins, although some soybean cultivars have coloured seed coats that contain these compounds. Here, we analyse the transcriptional control of PA and anthocyanin biosynthesis in soybean. Ectopic expression of the transcription factors (TFs) GmTT2A, GmTT2B, GmMYB5A or R in soybean hairy roots induced the accumulation of PAs (primarily in phloem tissues) or anthocyanins and led to up-regulation of 1775, 856, 1411 and 1766 genes, respectively, several of which encode enzymes involved in PA biosynthesis. The genes regulated by GmTT2A and GmTT2B partially overlapped, suggesting conserved but potentially divergent roles for these two TFs in regulating PA accumulation in soybean. The two key enzymes anthocyanidin reductase and leucoanthocyanidin reductase were differentially upregulated, by GmTT2A/GmTT2B and GmMYB5A, respectively. Transgenic soybean plants overexpressing GmTT2B or MtLAP1 (a proven up-regulator of the upstream reactions for production of precursors for PA biosynthesis in legumes) showed increased accumulation of PAs and anthocyanins, respectively, associated with transcriptional reprogramming paralleling the RNA-seq data collected in soybean hairy roots. Collectively, our results show that engineered PA biosynthesis in soybean exhibits qualitative and spatial differences from the better-studied model systems Arabidopsis thaliana and Medicago truncatula, and suggest targets for engineering PAs in soybean plants.

VvLAR1 and VvLAR2 Are Bifunctional Enzymes for Proanthocyanidin Biosynthesis in Grapevine.

Proanthocyanidins (PAs) in grapevine (Vitis vinifera) are found mainly in berries, and their content and degree of polymerization are important for the mouth feel of red wine. However, the mechanism of PA polymerization in grapevine remains unclear. Previous studies in the model legume Medicago truncatula showed that 4ß-(S-cysteinyl)-epicatechin (Cys-EC) is an epicatechin-type extension unit for nonenzymatic PA polymerization, and that leucoanthocyanidin reductase (LAR) converts Cys-EC into epicatechin starter unit to control PA extension. Grapevine possesses two LAR genes, but their functions are not clear. Here, we show that both Cys-EC and 4ß-(S-cysteinyl)-catechin (Cys-C) are present in grapevine. Recombinant VvLAR1 and VvLAR2 convert Cys-C and Cys-EC into (+)-catechin and (-)-epicatechin, respectively, in vitro. The kinetic parameters of VvLARs are similar, with both enzymes being more efficient with Cys-C than with Cys-EC, the 2,3-cis conformation of which results in steric hindrance in the active site. Both VvLARs also produce (+)-catechin from leucocyanidin, and an inactive VvLAR2 allele reported previously is the result of a single amino acid mutation in the N terminus critical for all NADPH-dependent activities of the enzyme. VvLAR1 or VvLAR2 complement the M. truncatula lar:ldox double mutant that also lacks the leucoanthocyanidin dioxygenase (LDOX) required for epicatechin starter unit formation, resulting in increased soluble PA levels, decreased insoluble PA levels, and reduced levels of Cys-C and Cys-EC when compared to the double mutant, and the appearance of catechin, epicatechin, and PA dimers characteristic of the ldox single mutant in young pods. These data advance our knowledge of PA building blocks and LAR function and provide targets for grapevine breeding to alter PA composition.

Proanthocyanidin subunit composition determined by functionally diverged dioxygenases.

Proanthocyanidins (PAs) are primarily composed of the flavan-3-ol subunits (-)-epicatechin and/or (+)-catechin, but the basis for their different starter and extension unit compositions remains unclear. Genetic and biochemical analyses show that, in the model legume Medicago truncatula, two 2-oxoglutarate-dependent dioxygenases, anthocyanidin synthase (ANS) and its homologue leucoanthocyanidin dioxygenase (LDOX), are involved in parallel pathways to generate, respectively, the (-)-epicatechin extension and starter units of PAs, with (+) catechin being an intermediate in the formation of the (-)-epicatechin starter unit. The presence/absence of the LDOX pathway accounts for natural differences in PA compositions across species, and engineering loss of function of ANS or LDOX provides a means to obtain PAs with different compositions and degrees of polymerization for use in food and feed.

Peptide signaling molecules CLE5 and CLE6 affect Arabidopsis leaf shape downstream of leaf patterning transcription factors and auxin.

Intercellular signaling mediated by small peptides is critical to coordinate organ formation in animals, but whether extracellular polypeptides play similar roles in plants is unknown. Here we describe a role in Arabidopsis leaf development for two members of the CLAVATA3/ESR-RELATED peptide family, CLE5 and CLE6, which lie adjacent to each other on chromosome 2. Uniquely among the CLE genes, CLE5 and CLE6 are expressed specifically at the base of developing leaves and floral organs, adjacent to the boundary with the shoot apical meristem. During vegetative development CLE5 and CLE6 transcription is regulated by the leaf patterning transcription factors BLADE-ON-PETIOLE1 (BOP1) and ASYMMETRIC LEAVES2 (AS2), as well as by the WUSCHEL-RELATED HOMEOBOX (WOX) transcription factors WOX1 and PRESSED FLOWER (PRS). Moreover, CLE5 and CLE6 transcript levels are differentially regulated in various genetic backgrounds by the phytohormone auxin. Analysis of loss-of-function mutations generated by genome engineering reveals that CLE5 and CLE6 independently and together have subtle effects on rosette leaf shape. Our study indicates that the CLE5 and CLE6 peptides function downstream of leaf patterning factors and phytohormones to modulate the final leaf morphology.

Programming of Plant Leaf Senescence with Temporal and Inter-Organellar Coordination of Transcriptome in Arabidopsis.

Plant leaves, harvesting light energy and fixing CO2, are a major source of foods on the earth. Leaves undergo developmental and physiological shifts during their lifespan, ending with senescence and death. We characterized the key regulatory features of the leaf transcriptome during aging by analyzing total- and small-RNA transcriptomes throughout the lifespan of Arabidopsis (Arabidopsis thaliana) leaves at multidimensions, including age, RNA-type, and organelle. Intriguingly, senescing leaves showed more coordinated temporal changes in transcriptomes than growing leaves, with sophisticated regulatory networks comprising transcription factors and diverse small regulatory RNAs. The chloroplast transcriptome, but not the mitochondrial transcriptome, showed major changes during leaf aging, with a strongly shared expression pattern of nuclear transcripts encoding chloroplast-targeted proteins. Thus, unlike animal aging, leaf senescence proceeds with tight temporal and distinct interorganellar coordination of various transcriptomes that would be critical for the highly regulated degeneration and nutrient recycling contributing to plant fitness and productivity.

The Transcriptional Repressor MYB2 Regulates Both Spatial and Temporal Patterns of Proanthocyandin and Anthocyanin Pigmentation in Medicago truncatula.

Accumulation of anthocyanins and proanthocyanidins (PAs) is limited to specific cell types and developmental stages, but little is known about how antagonistically acting transcriptional regulators work together to determine temporal and spatial patterning of pigmentation at the cellular level, especially for PAs. Here, we characterize MYB2, a transcriptional repressor regulating both anthocyanin and PA biosynthesis in the model legume Medicago truncatula. MYB2 was strongly upregulated by MYB5, a major regulator of PA biosynthesis in M. truncatula and a component of MYB-basic helix loop helix-WD40 (MBW) activator complexes. Overexpression of MYB2 abolished anthocyanin and PA accumulation in M. truncatula hairy roots and Arabidopsis thaliana seeds, respectively. Anthocyanin deposition was expanded in myb2 mutant seedlings and flowers accompanied by increased anthocyanin content. PA mainly accumulated in the epidermal layer derived from the outer integument in the M. truncatula seed coat, starting from the hilum area. The area of PA accumulation and ANTHOCYANIDIN REDUCTASE expression was expanded into the seed body at the early stage of seed development in the myb2 mutant. Genetic, biochemical, and cell biological evidence suggests that MYB2 functions as part of a multidimensional regulatory network to define the temporal and spatial pattern of anthocyanin and PA accumulation linked to developmental processes.

MYB5 and MYB14 Play Pivotal Roles in Seed Coat Polymer Biosynthesis in Medicago truncatula.

In Arabidopsis (Arabidopsis thaliana), the major MYB protein regulating proanthocyanidin (PA) biosynthesis is TT2, named for the transparent testa phenotype of tt2 mutant seeds that lack PAs in their coats. In contrast, the MYB5 transcription factor mainly regulates seed mucilage biosynthesis and trichome branching, with only a minor role in PA biosynthesis. We here characterize MYB5 and MYB14 (a TT2 homolog) in the model legume Medicago truncatula. Overexpression of MtMYB5 or MtMYB14 strongly induces PA accumulation in M. truncatula hairy roots, and both myb5 and myb14 mutants of M. truncatula exhibit darker seed coat color than wild-type plants, with myb5 also showing deficiency in mucilage biosynthesis. myb5 mutant seeds have a much stronger seed color phenotype than myb14. The myb5 and myb14 mutants accumulate, respectively, about 30% and 50% of the PA content of wild-type plants, and PA levels are reduced further in myb5 myb14 double mutants. Transcriptome analyses of overexpressing hairy roots and knockout mutants of MtMYB5 and MtMYB14 indicate that MtMYB5 regulates a broader set of genes than MtMYB14. Moreover, we demonstrate that MtMYB5 and MtMYB14 physically interact and synergistically activate the promoters of anthocyanidin reductase and leucoanthocyanidin reductase, the key structural genes leading to PA biosynthesis, in the presence of MtTT8 and MtWD40-1. Our results provide new insights into the complex regulation of PA and mucilage biosynthesis in M. truncatula.

Gene regulatory cascade of senescence-associated NAC transcription factors activated by ETHYLENE-INSENSITIVE2-mediated leaf senescence signalling in Arabidopsis.

Leaf senescence is a finely tuned and genetically programmed degeneration process, which is critical to maximize plant fitness by remobilizing nutrients from senescing leaves to newly developing organs. Leaf senescence is a complex process that is driven by extensive reprogramming of global gene expression in a highly coordinated manner. Understanding how gene regulatory networks involved in controlling leaf senescence are organized and operated is essential to decipher the mechanisms of leaf senescence. It was previously reported that the trifurcate feed-forward pathway involving EIN2, ORE1, and miR164 in Arabidopsis regulates age-dependent leaf senescence and cell death. Here, new components of this pathway have been identified, which enhances knowledge of the gene regulatory networks governing leaf senescence. Comparative gene expression analysis revealed six senescence-associated NAC transcription factors (TFs) (ANAC019, AtNAP, ANAC047, ANAC055, ORS1, and ORE1) as candidate downstream components of ETHYLENE-INSENSITIVE2 (EIN2). EIN3, a downstream signalling molecule of EIN2, directly bound the ORE1 and AtNAP promoters and induced their transcription. This suggests that EIN3 positively regulates leaf senescence by activating ORE1 and AtNAP, previously reported as key regulators of leaf senescence. Genetic and gene expression analyses in the ore1 atnap double mutant revealed that ORE1 and AtNAP act in distinct and overlapping signalling pathways. Transient transactivation assays further demonstrated that ORE1 and AtNAP could activate common as well as differential NAC TF targets. Collectively, the data provide insight into an EIN2-mediated senescence signalling pathway that coordinates global gene expression during leaf senescence via a gene regulatory network involving EIN3 and senescence-associated NAC TFs.

Metabolic engineering of anthocyanins and condensed tannins in plants.

Monomeric anthocyanins and polymeric proanthocyanidins (condensed tannins) contribute to important plant traits such as flower and fruit pigmentation, fruit astringency, disease resistance and forage quality. Recent advances in our understanding of the transcriptional control mechanisms that regulate anthocyanin and condensed tannin formation in plants suggest new approaches for the engineering of quality traits associated with these molecules. In particular, MYB family transcription factors are emerging as central players in the coordinated activation of sets of genes specific for the anthocyanin and tannin pathways. Mutations in these genes underlie potentially valuable crop traits, and ectopic over- or under-expression of MYB transcription factors provides routes for engineering of these complex pathways.

Shoot apical meristem form and function.

The shoot apical meristem (SAM) generates above-ground aerial organs throughout the lifespan of higher plants. In order to fulfill this function, the meristem must maintain a balance between the self-renewal of a reservoir of central stem cells and organ initiation from peripheral cells. The activity of the pluripotent stem cell population in the SAM is dynamically controlled by complex, overlapping signaling networks that include the feedback regulation of meristem maintenance genes and the signaling of plant hormones. Organ initiation likewise requires the function of multifactor gene regulatory networks, as well as instructive cues from the plant hormone auxin and reciprocal signals from the shoot meristem. Floral meristems (FMs) are products of the reproductive SAM that sustains a transient stem cell reservoir for flower formation. Regulation of FM activity involves both feedback loops shared with the SAM and floral-specific factors. Recent studies have rapidly advanced our understanding of SAM function by adopting newly developed molecular and computational techniques. These advances are becoming integrated with data from traditional molecular genetics methodologies to develop a framework for understanding the central principles of SAM function.

Analyzing shoot apical meristem development.

The shoot apical meristem of Arabidopsis thaliana contains a reservoir of pluripotent stem cells that functions as a continuous source of new cells for organ formation during development. The SAM forms during embryogenesis, when it becomes stratified into specific cell layers and zones that can be delineated based on morphological and molecular criteria. The primary SAM produces all the aerial structures of the adult plant, and alterations in SAM organization or function can have profound effects on vegetative and reproductive plant morphology. Such SAM-specific defects can be identified, evaluated, and quantified using specialized microscopic and histological techniques.

Control of Arabidopsis leaf morphogenesis through regulation of the YABBY and KNOX families of transcription factors.

The patterning of initiating organs along specific axes of polarity is critical for the proper development of all higher organisms. Plant lateral organs, such as leaves, are derived from the shoot apical meristems located at the growing tips. After initiation, the leaf primordia of species such as Arabidopsis thaliana differentiate into a polarized structure consisting of a proximal petiole and a distal blade, but the molecular mechanisms that control proximal-distal pattern formation are poorly understood. The transcriptional activators BLADE-ON-PETIOLE1 (BOP1) and BOP2 are known to control Arabidopsis lateral organ differentiation by regulating gene expression along the adaxial-abaxial (dorsal-ventral) and proximal-distal polarity axes. Here, we demonstrate that the development of ectopic blade tissue along bop1 bop2 leaf petioles is strongly suppressed in a dosage-dependant manner by mutations in either of two closely related YABBY (YAB) genes, FILAMENTOUS FLOWER (FIL) and YAB3. Three KNOTTED-LIKE HOMEOBOX (KNOX1) genes also make lesser, and partially redundant, contributions to ectopic blade development in bop1 bop2 leaves. Mutation of these YAB and KNOX1 genes together causes nearly complete suppression of bop1 bop2 ectopic organ outgrowth at the morphological and cellular levels. Our data demonstrate that BOP1 and BOP2 regulate leaf patterning by controlling YAB and KNOX1 gene activity in the developing petiole.

BLADE-ON-PETIOLE1 coordinates organ determinacy and axial polarity in arabidopsis by directly activating ASYMMETRIC LEAVES2.

Continuous organ formation is a hallmark of plant development that requires organ-specific gene activity to establish determinacy and axial patterning, yet the molecular mechanisms that coordinate these events remain poorly understood. Here, we show that the organ-specific BTB-POZ domain proteins BLADE-ON-PETIOLE1 (BOP1) and BOP2 function as transcriptional activators during Arabidopsis thaliana leaf formation. We identify as a direct target of BOP1 induction the ASYMMETRIC LEAVES2 (AS2) gene, which promotes leaf cell fate specification and adaxial polarity. We find that BOP1 associates with the AS2 promoter and that BOP1 and BOP2 are required for AS2 activation specifically in the proximal, adaxial region of the leaf, demonstrating a role for the BOP proteins as proximal-distal as well as adaxial-abaxial patterning determinants. Furthermore, repression of BOP1 and BOP2 expression by the indeterminacy-promoting KNOX gene SHOOTMERISTEMLESS is critical to establish a functional embryonic shoot apical meristem. Our data indicate that direct activation of AS2 transcription by BOP1 and BOP2 is vital for generating the conditions for KNOX repression at the leaf base and may represent a conserved mechanism for coordinating leaf morphogenesis with patterning along the adaxial-abaxial and the proximal-distal axes.

BLADE-ON-PETIOLE 1 and 2 control Arabidopsis lateral organ fate through regulation of LOB domain and adaxial-abaxial polarity genes.

We report a novel function for BLADE-ON-PETIOLE1 (BOP1) and BOP2 in regulating Arabidopsis thaliana lateral organ cell fate and polarity, through the analysis of loss-of-function mutants and transgenic plants that ectopically express BOP1 or BOP2. 35S:BOP1 and 35S:BOP2 plants exhibit a very short and compact stature, hyponastic leaves, and downward-orienting siliques. We show that the LATERAL ORGAN BOUNDARIES (LOB) domain genes ASYMMETRIC LEAVES2 (AS2) and LOB are upregulated in 35S:BOP and downregulated in bop mutant plants. Ectopic expression of BOP1 or BOP2 also results in repression of class I knox gene expression. We further demonstrate a role for BOP1 and BOP2 in establishing the adaxial-abaxial polarity axis in the leaf petiole, where they regulate PHB and FIL expression and overlap in function with AS1 and AS2. Interestingly, during this study, we found that KANADI1 (KAN1) and KAN2 act to promote adaxial organ identity in addition to their well-known role in promoting abaxial organ identity. Our data indicate that BOP1 and BOP2 act in cells adjacent to the lateral organ boundary to repress genes that confer meristem cell fate and induce genes that promote lateral organ fate and polarity, thereby restricting the developmental potential of the organ-forming cells and facilitating their differentiation.

BLADE-ON-PETIOLE1 encodes a BTB/POZ domain protein required for leaf morphogenesis in Arabidopsis thaliana.

The BLADE-ON-PETIOLE1 (BOP1) gene of Arabidopsis thaliana is required for proper leaf morphogenesis. BOP1 regulates leaf differentiation in a proximal-distal manner, and represses the expression of three class I knotted-like homeobox (knox) genes during leaf formation. Utilizing a map-based approach, we identified the molecular nature of the BOP1 gene, which encodes a BTB/POZ domain protein with ankyrin repeats. BOP1 is a member of a small gene family in Arabidopsis that includes the disease resistance regulatory protein NPR1. Insertions in and around BOP1 cause distinct lesions in leaf morphogenesis, revealing complex regulation of the locus. BOP1 transcripts are initially detectable in embryos, where they specifically localize to the base of the developing cotyledons near the SAM. During vegetative development, BOP1 is expressed in young leaf primordia and at the base of the rosette leaves on the adaxial side. During reproductive development, BOP1 transcripts are detected in young floral buds, and at the base of the sepals and petals. Our results indicate that BOP1 encodes a putative regulatory protein that modulates meristematic activity at discrete locations in developing lateral organs. This is the first report on a plant protein that plays a key role in morphogenesis with the distinctive combinatorial architecture of the BTB/POZ and ankyrin repeat domains.