Kengo Morohashi

The TTG1-bHLH-MYB complex controls trichome cell fate and patterning through direct targeting of regulatory loci

A network of three classes of proteins consisting of bHLH and MYB transcription factors, and a WD40 repeat protein, TRANSPARENT TESTA GLABRA1 (TTG1), act in concert to activate trichome initiation and patterning. Using YFP-TTG1 translational fusions, we show that TTG1 is expressed ubiquitously in Arabidopsis leaves and is preferentially localized in the nuclei of trichomes at all developmental stages. Using a conditional transgenic allele, we demonstrate that TTG1 directly targets the same genes as the bHLH protein GLABRA3 (GL3). In vivo binding of the R2R3-MYB protein GLABRA1 (GL1) to the promoters of GLABRA2 (GL2), TRANSPARENT TESTA GLABRA2 (TTG2), CAPRICE (CPC) and ENHANCER OF TRIPTYCHON AND CAPRICE1 (ETC1) establishes that these genes are major transcriptional targets for the TTG1-bHLH-MYB regulatory complex. By co-precipitation, we confirm that TTG1 associates with GL3 and GL1 in vivo, forming a complex. The loss of TTG1 and GL1 through mutation, affects the subcellular distribution of GL3. Using particle bombardment, we show that TTG1, GL3, GL1 and the homeodomain protein GL2 do not move between adjacent epidermal cells, while the R3-MYB, CPC, does move to neighboring cells. These data support a model for the TTG1 complex directly regulating activators and repressors and the movement of repressors to affect trichome patterning on the Arabidopsis leaf.

A systems approach reveals regulatory circuitry for Arabidopsis trichome initiation by the GL3 and GL1 selectors.

Position-dependent cell fate determination and pattern formation are unique aspects of the development of plant structures. The establishment of single-celled leaf hairs (trichomes) from pluripotent epidermal (protodermal) cells in Arabidopsis provides a powerful system to determine the gene regulatory networks involved in cell fate determination. To obtain a holistic view of the regulatory events associated with the differentiation of Arabidopsis epidermal cells into trichomes, we combined expression and genome-wide location analyses (ChIP-chip) on the trichome developmental selectors GLABRA3 (GL3) and GLABRA1 (GL1), encoding basic helix-loop-helix (bHLH) and MYB transcription factors, respectively. Meta-analysis was used to integrate genome-wide expression results contrasting wild type and gl3 or gl1 mutants with changes in gene expression over time using inducible versions of GL3 and GL1. This resulted in the identification of a minimal set of genes associated with the differentiation of epidermal cells into trichomes. ChIP-chip experiments, complemented by the targeted examination of factors known to participate in trichome initiation or patterning, identified about 20 novel GL3/GL1 direct targets. In addition to genes involved in the control of gene expression, such as the transcription factors SCL8 and MYC1, we identified SIM (SIAMESE), encoding a cyclin-dependent kinase inhibitor, and RBR1 (RETINOBLASTOMA RELATED1), corresponding to a negative regulator of the cell cycle transcription factor E2F, as GL3/GL1 immediate targets, directly implicating these trichome regulators in the control of the endocycle. The expression of many of the identified GL3/GL1 direct targets was specific to very early stages of trichome initiation, suggesting that they participate in some of the earliest known processes associated with protodermal cell differentiation. By combining this knowledge with the analysis of genes associated with trichome formation, our results reveal the architecture of the top tiers of the hierarchical structure of the regulatory network involved in epidermal cell differentiation and trichome formation.

Participation of the Arabidopsis bHLH Factor GL3 in Trichome Initiation Regulatory Events

The development of trichomes (leaf hairs) from pluripotent epidermal cells in Arabidopsis (Arabidopsis thaliana) provides a powerful system to investigate the regulatory motifs involved in plant cell differentiation. We show here that trichome initiation is triggered within 4 h of the induction of the GLABRA3 (GL3) basic helix-loop-helix transcription factor. Within this developmental window, GL3 binds to the promoters of at least three genes previously implicated in the development and patterning of trichomes (GL2, CAPRICE, and ENHANCER OF TRIPTYCHON AND CAPRICE1) and activates their transcription. The in vivo binding of GL3 to the promoters of these genes requires the presence of the R2R3-MYB factor GL1, supporting a model in which a GL3-GL1 complex is part of the trichome initiation enhanceosome. In contrast, GL3 is recruited to its own promoter in a GL1-independent manner, and this results in decreased GL3 expression, suggesting the presence of a GL3 negative autoregulatory loop. In support of genetic analyses indicating that ENHANCER OF GL3 (EGL3) is partially redundant with GL3, we show that EGL3 shares some direct targets with GL3. However, our results suggest that GL3 and EGL3 work independently of each other. Taken together, our results provide a regulatory framework to understand early events of epidermal cell differentiation.

Unraveling the KNOTTED1 regulatory network in maize meristems

KNOTTED1 (KN1)-like homeobox (KNOX) transcription factors function in plant meristems, self-renewing structures consisting of stem cells and their immediate daughters. We defined the KN1 cistrome in maize inflorescences and found that KN1 binds to several thousand loci, including 643 genes that are modulated in one or multiple tissues. These KN1 direct targets are strongly enriched for transcription factors (including other homeobox genes) and genes participating in hormonal pathways, most significantly auxin, demonstrating that KN1 plays a key role in orchestrating the upper levels of a hierarchical gene regulatory network that impacts plant meristem identity and function.

Regulation of Cell Proliferation in the Stomatal Lineage by the Arabidopsis MYB FOUR LIPS via Direct Targeting of Core Cell Cycle Genes

The MYB protein FOUR LIPS (FLP) promotes Arabidopsis stomatal patterning by suppressing cell division before differentiation. FLP direct targets were found to be enriched in cell cycle genes that function in both S-G1 and G2-M phase, indicating that this transcription factor acts as a developmental integrator. Stomata, which are epidermal pores surrounded by two guard cells, develop from a specialized stem cell lineage and function in shoot gas exchange. The Arabidopsis thaliana FOUR LIPS (FLP) and MYB88 genes encode closely related and atypical two-MYB-repeat proteins, which when mutated result in excess divisions and abnormal groups of stomata in contact. Consistent with a role in transcription, we show here that FLP and MYB88 are nuclear proteins with DNA binding preferences distinct from other known MYBs. To identify possible FLP/MYB88 transcriptional targets, we used chromatin immunoprecitation (ChIP) followed by hybridization to Arabidopsis whole genome tiling arrays. These ChIP-chip data indicate that FLP/MYB88 target the upstream regions especially of cell cycle genes, including cyclins, cyclin-dependent kinases (CDKs), and components of the prereplication complex. In particular, we show that FLP represses the expression of the mitosis-inducing factor CDKB1;1, which, along with CDKB1;2, is specifically required both for the last division in the stomatal pathway and for cell overproliferation in flp mutants. We propose that FLP and MYB88 together integrate patterning with the control of cell cycle progression and terminal differentiation through multiple and direct cell cycle targets. FLP recognizes a distinct cis-regulatory element that overlaps with that of the cell cycle activator E2F-DP in the CDKB1;1 promoter, suggesting that these MYBs may also modulate E2F-DP pathways.

A Genome-Wide Regulatory Framework Identifies Maize Pericarp Color1 Controlled Genes

This study combines high-throughput RNA sequencing and chromatin immunoprecipitation and sequencing to examine the effect of Pericarp Color1 (P1) on global gene expression in maize pericarps and silks, finding that, in addition to regulating flavonoid biosynthesis genes, P1 modulates the expression of a much larger gene set involved in primary metabolism and production of other specialized compounds. 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.

Molecular basis for the action of a dietary flavonoid revealed by the comprehensive identification of apigenin human targets

Significance The beneficial health effects of dietary phytochemicals make them promising candidates for treatment and prevention of multiple diseases. However, cellular targets for dietary components remain largely unknown. By combining phage display with high-throughput sequencing, we identified 160 human targets of apigenin, a flavonoid abundant in fruits and vegetables. The apigenin targets include hnRNPA2, a factor associated with numerous cellular malignancies and involved in mRNA metabolism/splicing. We show that, by inhibiting hnRNPA2 dimerization, apigenin affects the alternative splicing of key mRNAs. These findings provide a perspective on how dietary phytochemicals function and what distinguishes their action from pharmaceutical drugs. Flavonoids constitute the largest class of dietary phytochemicals, adding essential health value to our diet, and are emerging as key nutraceuticals. Cellular targets for dietary phytochemicals remain largely unknown, posing significant challenges for the regulation of dietary supplements and the understanding of how nutraceuticals provide health value. Here, we describe the identification of human cellular targets of apigenin, a flavonoid abundantly present in fruits and vegetables, using an innovative high-throughput approach that combines phage display with second generation sequencing. The 160 identified high-confidence candidate apigenin targets are significantly enriched in three main functional categories: GTPase activation, membrane transport, and mRNA metabolism/alternative splicing. This last category includes the heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2), a factor involved in splicing regulation, mRNA stability, and mRNA transport. Apigenin binds to the C-terminal glycine-rich domain of hnRNPA2, preventing hnRNPA2 from forming homodimers, and therefore, it perturbs the alternative splicing of several human hnRNPA2 targets. Our results provide a framework to understand how dietary phytochemicals exert their actions by binding to many functionally diverse cellular targets. In turn, some of them may modulate the activity of a large number of downstream genes, which is exemplified here by the effects of apigenin on the alternative splicing activity of hnRNPA2. Hence, in contrast to small-molecule pharmaceuticals designed for defined target specificity, dietary phytochemicals affect a large number of cellular targets with varied affinities that, combined, result in their recognized health benefits.

Cloning and characterization of a UV-B-inducible maize flavonol synthase

Flavonols 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.

Regulatory modules controlling maize inflorescence architecture

Genetic control of branching is a primary determinant of yield, regulating seed number and harvesting ability, yet little is known about the molecular networks that shape grain-bearing inflorescences of cereal crops. Here, we used the maize (Zea mays) inflorescence to investigate gene networks that modulate determinacy, specifically the decision to allow branch growth. We characterized developmental transitions by associating spatiotemporal expression profiles with morphological changes resulting from genetic perturbations that disrupt steps in a pathway controlling branching. Developmental dynamics of genes targeted in vivo by the transcription factor RAMOSA1, a key regulator of determinacy, revealed potential mechanisms for repressing branches in distinct stem cell populations, including interactions with KNOTTED1, a master regulator of stem cell maintenance. Our results uncover discrete developmental modules that function in determining grass-specific morphology and provide a basis for targeted crop improvement and translation to other cereal crops with comparable inflorescence architectures.

The basic helix–loop–helix domain of maize R links transcriptional regulation and histone modifications by recruitment of an EMSY-related factor

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.