SUMMARYThe expansion of gene families encoding regulatory proteins is typically associated with the increase in complexity characteristic of multi-cellular organisms. The MYB and basic helix-loop-helix (bHLH) families provide excellent examples of how gene duplication and divergence within particular groups of transcription factors are associated with, if not driven by, the morphological and metabolic diversity that characterize the higher plants. These gene families expanded dramatically in higher plants; for example, there are approximately 339 and 162 MYB and bHLH genes, respectively, in Arabidopsis, and approximately 230 and 111, respectively, in rice. In contrast, the Chlamydomonas genome has only 38 MYB genes and eight bHLH genes. In this review, we compare the MYB and bHLH gene families from structural, evolutionary and functional perspectives. The knowledge acquired on the role of many of these factors in Arabidopsis provides an excellent reference to explore sequence-function relationships in crops and other plants. The physical interaction and regulatory synergy between particular sub-classes of MYB and bHLH factors is perhaps one of the best examples of combinatorial plant gene regulation. However, members of the MYB and bHLH families also interact with a number of other regulatory proteins, forming complexes that either activate or repress the expression of sets of target genes that are increasingly being identified through a diversity of high-throughput genomic approaches. The next few years are likely to witness an increasing understanding of the extent to which conserved transcription factors participate at similar positions in gene regulatory networks across plant species.

Plants produce a very large number of specialized compounds that must be transported from their site of synthesis to the sites of storage or disposal. Anthocyanin accumulation has provided a powerful system to elucidate the molecular and cellular mechanisms associated with the intracellular trafficking of phytochemicals. Benefiting from the unique fluorescent properties of anthocyanins, we show here that in Arabidopsis (Arabidopsis thaliana), one route for anthocyanin transport to the vacuole involves vesicle-like structures shared with components of the secretory pathway. By colocalizing the red fluorescence of the anthocyanins with green fluorescent protein markers of the endomembrane system in Arabidopsis seedlings, we show that anthocyanins are also sequestered to the endoplasmic reticulum and to endoplasmic reticulum-derived vesicle-like structures targeted directly to the protein storage vacuole in a Golgi-independent manner. Moreover, our results indicate that vacuolar accumulation of anthocyanins does not depend solely on glutathione S-transferase activity or ATP-dependent transport mechanisms. Indeed, we observed a dramatic increase of anthocyanin-filled subvacuolar structures, without a significant effect on total anthocyanin levels, when we inhibited glutathione S-transferase activity, or the ATP-dependent transporters with vanadate, a general ATPase inhibitor. Taken together, these results provide evidence for an alternative novel mechanism of vesicular transport and vacuolar sequestration of anthocyanins in Arabidopsis.

Pericarp Color1 (P1) encodes an R2R3-MYB transcription factor responsible for the accumulation of insecticidal flavones in maize (Zea mays) silks and red phlobaphene pigments in pericarps and other floral tissues, which makes P1 an important visual marker. Using genome-wide expression analyses (RNA sequencing) in pericarps and silks of plants with contrasting P1 alleles combined with chromatin immunoprecipitation coupled with high-throughput sequencing, we show here that the regulatory functions of P1 are much broader than the activation of genes corresponding to enzymes in a branch of flavonoid biosynthesis. P1 modulates the expression of several thousand genes, and ;1500 of them were identified as putative direct targets of P1. Among them, we identified F2H1, corresponding to a P450 enzyme that converts naringenin into 2-hydroxynaringenin, a key branch point in the P1-controlled pathway and the first step in the formation of insecticidal C-glycosyl flavones. Unexpectedly, the binding of P1 to gene regulatory regions can result in both gene activation and repression. Our results indicate that P1 is the major regulator for a set of genes involved in flavonoid biosynthesis and a minor modulator of the expression of a much larger gene set that includes genes involved in primary metabolism and production of other specialized compounds.

The maize basic-helix-loop-helix (bHLH) factor R belongs to a group of proteins with important functions in the regulation of metabolism and development through the cooperation with R2R3-MYB transcription factors. Here we show that in addition to the bHLH and the R2R3-MYB-interacting domains, R contains a dimerization region located C-terminal to the bHLH motif. This protein-protein interaction domain is important for the regulation of anthocyanin pigment biosynthesis by contributing to the recruitment of the C1 R2R3-MYB factor to the C1 binding sites present in the promoters of flavonoid biosynthetic genes. The R dimerization region bares structural similarity to the ACT domain present in several metabolic enzymes. Protein fold recognition analyses resulted in the identification of similar ACT-like domains in several other plant bHLH proteins. We show that at least one of these related motifs is capable of mediating homodimer formation. These findings underscore the function of R as a docking site for multiple protein-protein interactions and provide evidence for the presence of a novel dimerization domain in multiple plant bHLH proteins.Proteins containing the basic-helix-loop-helix (bHLH) 3 domain compose one of the largest transcription factor families in plants (1-4). The bHLH signature that defines the family is constituted by an N-terminal ϳ16-amino acid-long basic ␣-helix that binds DNA to the canonical E-box (CANNTG) (5) and a C-terminal helix-loop-helix (HLH) domain involved in homo-and/or heterodimerization (6). Some factors, however, such as the Id myogenic regulator, lack the basic region and function as inhibitors by forming heterodimers that cannot bind DNA (7). It is common for bHLH proteins to contain additional protein-protein interaction domains that contribute in unique ways to their regulatory function (8). For example, the Myc proto-oncoprotein forms heterodimers with Max through the respective bHLH and adjacent basic leucine zipper (bZip) domains (9). In contrast to Myc, which cannot homodimerize, Max can form homo-or heterodimers with several related proteins, including Mad1 and Mnt (10). The bHLH region of Myc mediates the interaction with Miz-1, a POZ transcription factor that permits Myc to bind and repress promoters lacking the CACGTG E-box (10, 11). Plant bHLH proteins are also characterized by the presence of several conserved domains in addition to the bHLH motif. For example, the analysis of the 133 Arabidopsis bHLH factors uncovered 40 or more domains present in three or more proteins (3). By and large the function of these domains is not known. Among the few for which functions have been identified are the N-terminally located small APB domain present in several phytochrome-interacting factors (12) and the region that mediates the interaction with R2R3-MYB factors (13, 14) central to providing R2R3-MYB transcriptional regulators with very similar DNA binding preferences with the ability to control distinct sets of target genes in vivo (15).The bHLH/R2R3-MYB cooperation is best e...

The maize R2R3-MYB regulator C1 cooperates with the basic helixloop-helix (bHLH) factor R to activate the expression of anthocyanin biosynthetic genes coordinately. As is the case for other bHLH factors, R harbors several protein-protein interaction domains. Here we show that not the classical but rather a briefly extended R bHLH region forms homodimers that bind canonical G-box DNA motifs. This bHLH DNA-binding activity is abolished if the C-terminal ACT (aspartokinase, chorismate, and TyrA) domain is licensed to homodimerize. Then the bHLH remains in the monomeric form, allowing it to interact with R-interacting factor 1 (RIF1). In this configuration, the R-RIF1 complex is recruited to the promoters of a subset of anthocyanin biosynthetic genes, such as A1, through the interaction with its MYB partner C1. If, however, the ACT domain remains monomeric, the bHLH region dimerizes and binds to G-boxes present in several anthocyanin genes, such as Bz1. Our results provide a mechanism by which a dimerization domain in a bHLH factor behaves as a switch that permits distinct configurations of a regulatory complex to be tethered to different promoters. Such a combinatorial gene regulatory framework provides one mechanism by which genes lacking obviously conserved cis-regulatory elements are regulated coordinately.gene regulation | promoter switch T he evolution of multicellular organisms was accompanied by an increase in the complexity of gene-regulatory mechanisms, reflected in the expansion of transcription factor families and in the intricacy of the interactions between regulatory proteins and cis-regulatory elements in what is known today as "combinatorial transcriptional control." A premise of combinatorial control is that different arrangements of a discrete number of regulatory proteins can be used to regulate a much larger number of genes. Therefore, understanding how interactions between different regulatory proteins impact their ability to deploy the expression of specific gene sets is of fundamental biological importance.The basic helix-loop-helix (bHLH) family of transcription factors is among the largest in multicellular organisms (1). The hallmark of the family is a bHLH domain, which consists of two functionally distinct regions. Generally, the basic region of the bHLH domain directly contacts DNA harboring an E-box sequence (CANNTG), and the HLH region provides the potential for homo-and heterodimerization. In addition to the HLH motif, bHLH factors often contain additional protein-protein interaction domains. For example, members of the MYC family of mammalian cell proliferation regulators, such as MAD or MNT, contain a leucine-zipper (LZ) region that contributes to the selective interaction with MAX, another bHLH-LZ protein (2). MAX can form homo-or heterodimers with several related proteins, including MAD (3) and MNT (4). Providing a textbook example of combinatorial transcriptional control, MYC-MAX and MAX-MAX complexes bind E-boxes, but only the MYC-MAX heterodimer activates cell-proliferation gene...

SUMMARYFlavonols are important compounds for conditional male fertility in maize (Zea mays) and other crops, and they also contribute to protecting plants from UV-B radiation. However, little continues to be known on how maize and other grasses synthesize flavonols, and how flavonol biosynthesis is regulated. By homology with an Arabidopsis flavonol synthase (AtFLS1), we cloned a maize gene encoding a protein (ZmFLS1) capable of converting the dihydrokaempferol (DHK) and dihydroquercetin (DHQ) dihydroflavonols to the corresponding flavonols, kaempferol (K) and quercetin (Q). Moreover, ZmFLS1 partially complements the flavonol deficiency of the Arabidopsis fls1 mutant, and restores anthocyanin accumulation to normal levels. We demonstrate that ZmFLS1 is under the control of the anthocyanin (C1/PL1 + R/B) and 3-deoxy flavonoid (P1) transcriptional regulators. Indeed, using chromatin immunoprecipitation (ChIP) experiments, we establish that ZmFLS1 is an immediate direct target of the P1 and C1/R regulatory complexes, revealing similar control as for earlier steps in the maize flavonoid pathway. Highlighting the importance of flavonols in UV-B protection, we also show that ZmFLS1 is induced in maize seedlings by UV-B, and that this induction is in part mediated by the increased expression of the P1, B and PL1 regulators. Together, our results identify a key flavonoid biosynthetic enzyme so far missed in maize and other monocots, and illustrate mechanisms by which flavonol accumulation is controlled in maize.

The R2R3 MYB transcription factor C1 requires the basic helix-loop-helix factor R as an essential co-activator for the transcription of maize anthocyanin genes. In contrast, the R2R3 MYB protein P1 activates a subset of the C1-regulated genes independently of R. Substitution of six amino acids in P1 with the C1 amino acids results in P1*, whose activity on C1-regulated and P1-regulated genes is R-dependent or R-enhanced, respectively. We have used P1* in combination with various promoters to uncover two mechanisms for R function. On synthetic promoters that contain only C1/P1 binding sites, R is an essential co-activator of C1. This function of R is unlikely to simply be the result of an increase in the C1 DNA-binding affinity, since transcriptional activity of a C1 mutant that binds DNA at a higher affinity, comparable with P1, remains R-dependent. The differential transcriptional activity of C1 fusions with the yeast Gal4 DNA-binding domain in yeast and maize cells suggests that part of the function of R is to relieve C1 from a plant-specific inhibitor. A second function of R requires cis-regulatory elements in addition to the C1/P1 DNAbinding sites for R-enhanced transcription of a1. We hypothesize that R functions in this mode by binding or recruiting additional factors to the anthocyanin regulatory element conserved in the promoters of several anthocyanin genes. Together, these findings suggest a model in which combinatorial interactions with co-activators enable R2R3 MYB factors with very similar DNA binding preferences to discriminate between target genes in vivo.Flowering plants express a large number of proteins containing the conserved R2R3 MYB DNA-binding domain. About 125 R2R3 Myb genes are present in the Arabidopsis genome (1), and many more are predicted to be expressed in maize and related monocots (2, 3). Similar to other transcription factor families, the R2R3 MYB factors show exquisite regulatory specificity in vivo, while recognizing very similar DNA sequences in vitro (4 -9). Thus, mechanisms other than discrimination between similar DNA-binding sites are at play in the control of specific sets of target genes by each R2R3 MYB transcription factor in vivo.The regulation of flavonoid biosynthetic gene expression by the cooperation of R2R3 MYB and basic helix-loop-helix (bHLH)1 transcription factors provides one of the best described examples of combinatorial gene regulation in plants (10,11). Anthocyanin accumulation in maize is controlled by two classes of regulatory proteins that act in concert: C1 or PL1, two closely related R2R3 MYB domain proteins (12), and R or B, which are members of the R/B family of bHLH domain proteins (13). Extensive genetic and molecular studies have shown that the C1 or Pl1 genes require a member of the bHLH-containing R or B gene family to activate transcription of the anthocyanin biosynthetic genes (10). The C1-and R/Bencoded proteins physically interact, and this interaction is mediated by the MYB domain of C1 and the N-terminal region of B (14) or R (15).The m...

The control of anthocyanin accumulation in maize by the cooperation of the basic helix-loop-helix (bHLH) protein R with the MYB transcription factor C1 provides one of the best examples of plant combinatorial transcriptional control. Establishing the function of the bHLH domain of R has remained elusive, and so far no proteins that interact with this conserved domain have been identified. We show here that the bHLH domain of R is dispensable for the activation of transiently expressed genes yet is essential for the activation of the endogenous genes in their normal chromatin environment. The activation of A1, one of the anthocyanin biosynthetic genes, is associated with increased acetylation of histone 3 (H3) at K9/K14 in the promoter region to which the C1/R complex binds. We identified R-interacting factor 1 (RIF1) as a nuclear, AGENET domain-containing EMSY-like protein that specifically interacts with the bHLH region of R. Knockdown experiments show that RIF1 is necessary for the activation of the endogenous promoters but not of transiently expressed genes. ChIP experiments established that RIF1 is tethered to the regulatory region of the A1 promoter by the C1/R complex. Together, these findings describe a function for the bHLH domain of R in linking transcriptional regulation with chromatin functions by the recruitment of an EMSY-related factor.anthocyanin ͉ BRCA2 ͉ chromatin T he evolution of multicellular organisms was accompanied by an increase in the complexity of gene regulatory mechanisms, reflected in the dramatic expansion of transcription factor families and in the intricate interactions between regulatory proteins and cis-regulatory elements in what is commonly known as combinatorial transcriptional control. Superimposed on this complexity is the understanding that histone modifications and chromatin structure are intimately linked to the regulatory activity of many transcription factors (1). Establishing the interactions between combinatorial gene regulation and histone functions thus poses a problem of significant biological importance.The basic helix-loop-helix (bHLH) family of transcription factors is among the largest in animals and plants (2). bHLH domains are characterized by the presence of an Ϸ18-residue hydrophilic basic helix followed by two amphipathic ␣-helices separated by a loop (3, 4). When present, basic regions contribute to the binding of bHLH factors to DNA, through cisregulatory elements, termed E-boxes, with the CANNTG consensus, whereas HLH motifs participate in homodimer or heterodimer formation (4). Maize R was the first plant bHLH transcription factor described (5). R belongs to a small gene family, which includes B, and R/B specify anthocyanin pigmentation in different plant tissues (6). They participate in the transcriptional regulation of the anthocyanin pathway genes through the cooperation with the R2R3-MYB transcription factor C1 or its paralog, PL1 (7). C1 and R/B physically interact through the MYB domain of C1 and the N-terminal region of R (which does not contai...