Nobutoshi Yamaguchi

LEAFY Target Genes Reveal Floral Regulatory Logic, cis Motifs, and a Link to Biotic Stimulus Response

The transition from vegetative growth to flower formation is critical for the survival of flowering plants. The plant-specific transcription factor LEAFY (LFY) has central, evolutionarily conserved roles in this process, both in the formation of the first flower and later in floral patterning. We performed genome-wide binding and expression studies to elucidate the molecular mechanisms by which LFY executes these roles. Our study reveals that LFY directs an elaborate regulatory network in control of floral homeotic gene expression. LFY also controls the expression of genes that regulate the response to external stimuli in Arabidopsis. Thus, our findings support a key role for LFY in the coordination of reproductive stage development and disease response programs in plants that may ensure optimal allocation of plant resources for reproductive fitness. Finally, motif analyses reveal a possible mechanism for stage-specific LFY recruitment and suggest a role for LFY in overcoming polycomb repression.

SWI2/SNF2 chromatin remodeling ATPases overcome polycomb repression and control floral organ identity with the LEAFY and SEPALLATA3 transcription factors.

Patterning of the floral organs is exquisitely controlled and executed by four classes of homeotic regulators. Among these, the class B and class C floral homeotic regulators are of central importance as they specify the male and female reproductive organs. Inappropriate induction of the class B gene APETALA3 (AP3) and the class C gene AGAMOUS (AG) causes reduced reproductive fitness and is prevented by polycomb repression. At the onset of flower patterning, polycomb repression needs to be overcome to allow induction of AP3 and AG and formation of the reproductive organs. We show that the SWI2/SNF2 chromatin-remodeling ATPases SPLAYED (SYD) and BRAHMA (BRM) are redundantly required for flower patterning and for the activation of AP3 and AG. The SWI2/SNF2 ATPases are recruited to the regulatory regions of AP3 and AG during flower development and physically interact with two direct transcriptional activators of class B and class C gene expression, LEAFY (LFY) and SEPALLATA3 (SEP3). SYD and LFY association with the AP3 and AG regulatory loci peaks at the same time during flower patterning, and SYD binding to these loci is compromised in lfy and lfy sep3 mutants. This suggests a mechanism for SWI2/SNF2 ATPase recruitment to these loci at the right stage and in the correct cells. SYD and BRM act as trithorax proteins, and the requirement for SYD and BRM in flower patterning can be overcome by partial loss of polycomb activity in curly leaf (clf) mutants, implicating the SWI2/SNF2 chromatin remodelers in reversal of polycomb repression.

Gibberellin Acts Positively Then Negatively to Control Onset of Flower Formation in Arabidopsis

One Hormone, Two Phases The switch from vegetative growth to flowering in the plant Arabidopsis involves two phases—inflorescence branching and flowering. Yamaguchi et al. (p. 638) examined how the phytohormone gibberellin regulates each phase differently. First, gibberellin levels increase and stimulate production of key flowering factors, one of which degrades gibberellin. As gibberellin levels then fall, the next phase of flowering factors is released from gibberellin repression. By regulating inflorescence branching separately from flowering, this system determines overall seed yield. Inflorescence architecture is shaped by a biphasic signaling network involving the plant hormone gibberellin. The switch to reproductive development is biphasic in many plants, a feature important for optimal pollination and yield. We show that dual opposite roles of the phytohormone gibberellin underpin this phenomenon in Arabidopsis. Although gibberellin promotes termination of vegetative development, it inhibits flower formation. To overcome this effect, the transcription factor LEAFY induces expression of a gibberellin catabolism gene; consequently, increased LEAFY activity causes reduced gibberellin levels. This allows accumulation of gibberellin-sensitive DELLA proteins. The DELLA proteins are recruited by SQUAMOSA PROMOTER BINDING PROTEIN–LIKE transcription factors to regulatory regions of the floral commitment gene APETALA1 and promote APETALA1 up-regulation and floral fate synergistically with LEAFY. The two opposing functions of gibberellin may facilitate evolutionary and environmental modulation of plant inflorescence architecture.

Auxin-regulated chromatin switch directs acquisition of flower primordium founder fate

Reprogramming of cell identities during development frequently requires changes in the chromatin state that need to be restricted to the correct cell populations. Here we identify an auxin hormone-regulated chromatin state switch that directs reprogramming from transit amplifying to primordium founder cell fate in Arabidopsis inflorescences. Upon auxin sensing, the MONOPTEROS transcription factor recruits SWI/SNF chromatin remodeling ATPases to increase accessibility of the DNA for induction of key regulators of flower primordium initiation. In the absence of the hormonal cue, auxin sensitive Aux/IAA proteins bound to MONOPTEROS block recruitment of the SWI/SNF chromatin remodeling ATPases in addition to recruiting a co-repressor/histone deacetylase complex. This simple and elegant hormone-mediated chromatin state switch is ideally suited for iterative flower primordium initiation and orchestrates additional auxin-regulated cell fate transitions. Our findings establish a new paradigm for nuclear response to auxin. They also provide an explanation for how this small molecule can direct diverse plant responses. DOI: http://dx.doi.org/10.7554/eLife.09269.001

LATE MERISTEM IDENTITY2 acts together with LEAFY to activate APETALA1

The switch from producing vegetative structures (branches and leaves) to producing reproductive structures (flowers) is a crucial developmental transition that significantly affects the reproductive success of flowering plants. In Arabidopsis, this transition is in large part controlled by the meristem identity regulator LEAFY (LFY). The molecular mechanisms by which LFY orchestrates a precise and robust switch to flower formation is not well understood. Here, we show that the direct LFY target LATE MERISTEM IDENTITY2 (LMI2) has a role in the meristem identity transition. Like LFY, LMI2 activates AP1 directly; moreover, LMI2 and LFY interact physically. LFY, LMI2 and AP1 are connected in a feed-forward and positive feedback loop network. We propose that these intricate regulatory interactions not only direct the precision of this crucial developmental transition in rapidly changing environmental conditions, but also contribute to its robustness and irreversibility.

PROTOCOLS: Chromatin Immunoprecipitation from Arabidopsis Tissues.

The ability of proteins to associate with genomic DNA in the context of chromatin is critical for many nuclear processes including transcription, replication, recombination, and DNA repair. Chromatin immunoprecipication (ChIP) is a practical and useful technique for characterizing protein / DNA association in vivo. The procedure generally includes six steps: (1) crosslinking the protein to the DNA; (2) isolating the chromatin; (3) chromatin fragmentation; (4) imunoprecipitation with antibodies against the protein of interest; (5) DNA recovery; and (6) PCR identification of factor associated DNA sequences. In this protocol, we describe guidelines, experimental setup, and conditions for ChIP in intact Arabidopsis tissues. This protocol has been used to study association of histone modifications, of chromatin remodeling ATPases, as well as of sequence-specific transcription factors with the genomic DNA in various Arabidopsis thaliana tissues. The protocol described focuses on ChIP-qPCR, but can readily be adapted for use in ChIP-chip or ChIP-seq experiments. The entire procedure can be completed within 3 days.

Mutations in two non-canonical Arabidopsis SWI2/SNF2 chromatin remodeling ATPases cause embryogenesis and stem cell maintenance defects

SWI2/SNF2 chromatin remodeling ATPases play important roles in plant and metazoan development. Whereas metazoans generally encode one or two SWI2/SNF2 ATPase genes, Arabidopsis encodes four such chromatin regulators: the well-studied BRAHMA and SPLAYED ATPases, as well as two closely related non-canonical SWI2/SNF2 ATPases, CHR12 and CHR23. No developmental role has as yet been described for CHR12 and CHR23. Here, we show that although strong single chr12 or chr23 mutants are morphologically indistinguishable from the wild type, chr12 chr23 double mutants cause embryonic lethality. The double mutant embryos fail to initiate root and shoot meristems, and display few and aberrant cell divisions. Weak double mutant embryos give rise to viable seedlings with dramatic defects in the maintenance of both the shoot and the root stem cell populations. Paradoxically, the stem cell defects are correlated with increased expression of the stem cell markers WUSCHEL and WOX5. During subsequent development, the meristem defects are partially overcome to allow for the formation of very small, bushy adult plants. Based on the observed morphological defects, we named the two chromatin remodelers MINUSCULE 1 and 2. Possible links between minu1 minu2 defects and defects in hormone signaling and replication-coupled chromatin assembly are discussed.

AINTEGUMENTA and AINTEGUMENTA-LIKE6/PLETHORA3 Induce LEAFY Expression in Response to Auxin to Promote the Onset of Flower Formation in Arabidopsis

Two parallel pathways confer floral identity on developing lateral primordia downstream of auxin. Proper timing of the onset to flower formation is critical for reproductive success. Monocarpic plants like Arabidopsis (Arabidopsis thaliana) switch from production of branches in the axils of leaves to that of flowers once in their lifecycle, during the meristem identity transition. The plant-specific transcription factor LEAFY (LFY) is necessary and sufficient for this transition. Previously, we reported that the plant hormone auxin induces LFY expression through AUXIN RESPONSE FACTOR5/MONOPTEROS (ARF5/MP). It is not known whether MP is solely responsible for auxin-directed transcriptional activation of LFY. Here, we show that two transcription factors belonging to the AINTEGUMENTA-LIKE/PLETHORA family, AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6/PLETHORA3 (AIL6/PLT3), act in parallel with MP to upregulate LFY in response to auxin. ant ail6 mutants display a delay in the meristem identity transition and in LFY induction. ANT and AIL6/PLT3 are expressed prior to LFY and bind to the LFY promoter to control LFY mRNA accumulation. Genetic and promoter/reporter studies suggest that ANT/AIL6 act in parallel with MP to promote LFY induction in response to auxin sensing. Our study highlights the importance of two separate auxin-controlled pathways in the meristem identity transition.

LEAFY controls Arabidopsis pedicel length and orientation by affecting adaxial–abaxial cell fate

Pedicel length and orientation (angle) contribute to the diversity of inflorescence architecture, and are important for optimal positioning of the flowers. However, relatively little is known about pedicel development. We previously described the Arabidopsis CORYMBOSA1 (CRM1)/BIG gene, which affects inflorescence architecture by controlling pedicel elongation and orientation. Here, we performed a suppressor screen using the partial loss-of-function allele crm1-13 to identify genes and pathways that affect pedicel development. We identified a hypomorph allele of the meristem identity regulator LEAFY (LFY) as the suppressor. Consistent with this, crm1 pedicels had elevated LFY levels and conditional gain of LFY function produced downward-bending pedicels. Steroid activation of 35S:LFY-GR plants caused a reduction in the cortical cell length in the abaxial domain and additional defects associated with adaxialization. Further analyses of loss of LFY function revealed that LFY is required for reduced cortical cell elongation at the adaxial side of the pedicel base. Defects in conditional LFY gain-of-function pedicels were correlated with decreased BREVIPEDICELLUS (BP) expression, while ASYMMETRIC LEAVES2 (AS2), a transcriptional repressor of BP, and REVOLUTA, a promoter of adaxial cell fate, were highly and ectopically expressed in LFY gain-of-function pedicels. LFY bound to cis-regulatory regions upstream of AS2, and as2 mutations partially suppressed the pedicel length and orientation defects caused by increased LFY activity. These data suggest that LFY activity promotes adaxial cell fate and hence the proper orientation and length of the pedicel partly by directly activating AS2 expression, which suppresses BP expression.

Transcriptional programs regulated by both LEAFY and APETALA1 at the time of flower formation

Two key regulators of the switch to flower formation and of flower patterning in Arabidopsis are the plant-specific helix-turn-helix transcription factor LEAFY (LFY) and the MADS box transcription factor APETALA1 (AP1). The interactions between these two transcriptional regulators are complex. AP1 is both a direct target of LFY and can act in parallel with LFY. Available genetic and molecular evidence suggests that LFY and AP1 together orchestrate the switch to flower formation and early events during flower morphogenesis by altering transcriptional programs. However, very little is known about target genes regulated by both transcription factors. Here, we performed a meta-analysis of public datasets to identify genes that are likely to be regulated by both LFY and AP1. Our analyses uncovered known and novel direct LFY and AP1 targets with a role in the control of onset of flower formation. It also identified additional families of proteins and regulatory pathways that may be under transcriptional control by both transcription factors. In particular, several of these genes are linked to response to hormones, to transport and to development. Finally, we show that the gibberellin catabolism enzyme ELA1, which was recently shown to be important for the timing of the switch to flower formation, is positively feedback-regulated by AP1. Our study contributes to the elucidation of the regulatory network that leads to formation of a vital plant organ system, the flower.