SHR and SCR coordinate root patterning and growth early in the cell cycle.

Precise control of cell division is essential for proper patterning and growth during the development of multicellular organisms. Coordination of formative divisions that generate new tissue patterns with proliferative divisions that promote growth is poorly understood. SHORTROOT (SHR) and SCARECROW (SCR) are transcription factors that are required for formative divisions in the stem cell niche of Arabidopsis roots1,2. Here we show that levels of SHR and SCR early in the cell cycle determine the orientation of the division plane, resulting in either formative or proliferative cell division. We used 4D quantitative, long-term and frequent (every 15 min for up to 48 h) light sheet and confocal microscopy to probe the dynamics of SHR and SCR in tandem within single cells of living roots. Directly controlling their dynamics with an SHR induction system enabled us to challenge an existing bistable model3 of the SHR-SCR gene-regulatory network and to identify key features that are essential for rescue of formative divisions in shr mutants. SHR and SCR kinetics do not align with the expected behaviour of a bistable system, and only low transient levels, present early in the cell cycle, are required for formative divisions. These results reveal an uncharacterized mechanism by which developmental regulators directly coordinate patterning and growth.

Tracking transcription factor mobility and interaction in Arabidopsis roots with fluorescence correlation spectroscopy.

To understand complex regulatory processes in multicellular organisms, it is critical to be able to quantitatively analyze protein movement and protein-protein interactions in time and space. During Arabidopsis development, the intercellular movement of SHORTROOT (SHR) and subsequent interaction with its downstream target SCARECROW (SCR) control root patterning and cell fate specification. However, quantitative information about the spatio-temporal dynamics of SHR movement and SHR-SCR interaction is currently unavailable. Here, we quantify parameters including SHR mobility, oligomeric state, and association with SCR using a combination of Fluorescent Correlation Spectroscopy (FCS) techniques. We then incorporate these parameters into a mathematical model of SHR and SCR, which shows that SHR reaches a steady state in minutes, while SCR and the SHR-SCR complex reach a steady-state between 18 and 24 hr. Our model reveals the timing of SHR and SCR dynamics and allows us to understand how protein movement and protein-protein stoichiometry contribute to development.

Transcriptional control of tissue formation throughout root development.

Tissue patterns are dynamically maintained. Continuous formation of plant tissues during postembryonic growth requires asymmetric divisions and the specification of cell lineages. We show that the BIRDs and SCARECROW regulate lineage identity, positional signals, patterning, and formative divisions throughout Arabidopsis root growth. These transcription factors are postembryonic determinants of the ground tissue stem cells and their lineage. Upon further activation by the positional signal SHORT-ROOT (a mobile transcription factor), they direct asymmetric cell divisions and patterning of cell types. The BIRDs and SCARECROW with SHORT-ROOT organize tissue patterns at all formative steps during growth, ensuring developmental plasticity.

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.

Identification of direct targets of plant transcription factors using the GR fusion technique.

The glucocorticoid receptor-dependent activation of plant transcription factors has proven to be a powerful tool for the identification of their direct target genes. In the absence of the synthetic steroid hormone dexamethasone (dex), transcription factors fused to the hormone-binding domain of the glucocorticoid receptor (TF-GR) are held in an inactive state, due to their cytoplasmic localization. This requires physical interaction with the heat shock protein 90 (HSP90) complex. Hormone binding leads to disruption of the interaction between GR and HSP90 and allows TF-GR fusion proteins to enter the nucleus. Once inside the nucleus, they bind to specific DNA sequences and immediately activate or repress expression of their targets. This system is well suited for the identification of direct target genes of transcription factors in plants, as (A) there is little basal protein activity in the absence of dex, (B) steroid application leads to rapid transcription factor activation, (C) no side effects of dex treatment are observed on the physiology of the plant, and (D) secondary effects of transcription factor activity can be eliminated by simultaneous application of an inhibitor of protein biosynthesis, cycloheximide (cyc). In this chapter, we describe detailed protocols for the preparation of plant material, for dex and cyc treatment, for RNA extraction, and for the PCR-based or genome-wide identification of direct targets of transcription factors fused to GR.

Paired-end analysis of transcription start sites in Arabidopsis reveals plant-specific promoter signatures.

Understanding plant gene promoter architecture has long been a challenge due to the lack of relevant large-scale data sets and analysis methods. Here, we present a publicly available, large-scale transcription start site (TSS) data set in plants using a high-resolution method for analysis of 5' ends of mRNA transcripts. Our data set is produced using the paired-end analysis of transcription start sites (PEAT) protocol, providing millions of TSS locations from wild-type Columbia-0 Arabidopsis thaliana whole root samples. Using this data set, we grouped TSS reads into "TSS tag clusters" and categorized clusters into three spatial initiation patterns: narrow peak, broad with peak, and weak peak. We then designed a machine learning model that predicts the presence of TSS tag clusters with outstanding sensitivity and specificity for all three initiation patterns. We used this model to analyze the transcription factor binding site content of promoters exhibiting these initiation patterns. In contrast to the canonical notions of TATA-containing and more broad "TATA-less" promoters, the model shows that, in plants, the vast majority of transcription start sites are TATA free and are defined by a large compendium of known DNA sequence binding elements. We present results on the usage of these elements and provide our Plant PEAT Peaks (3PEAT) model that predicts the presence of TSSs directly from sequence.

Gibberellin acts positively then negatively to control onset of flower formation in Arabidopsis.

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.

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.

LEAFY and Polar Auxin Transport Coordinately Regulate Arabidopsis Flower Development.

The plant specific transcription factor LEAFY (LFY) plays a pivotal role in the developmental switch to floral meristem identity in Arabidopsis. Our recent study revealed that LFY additionally acts downstream of AUXIN RESPONSE FACTOR5/MONOPTEROS to promote flower primordium initiation. LFY also promotes initiation of the floral organ and floral organ identity. To further investigate the interplay between LFY and auxin during flower development, we examined the phenotypic consequence of disrupting polar auxin transport in lfy mutants by genetic means. Plants with compromised LFY activity exhibit increased sensitivity to disruption of polar auxin transport. Compromised polar auxin transport activity in the lfy mutant background resulted in formation of fewer floral organs, abnormal gynoecium development, and fused sepals. In agreement with these observations, expression of the auxin response reporter DR5rev::GFP as well as of the direct LFY target CUP-SHAPED COTYLEDON2 were altered in lfy mutant flowers. We also uncovered reduced expression of ETTIN, a regulator of gynoecium development and a direct LFY target. Our results suggest that LFY and polar auxin transport coordinately modulate flower development by regulating genes required for elaboration of the floral organs.

A molecular framework for auxin-mediated initiation of flower primordia.

A classical role of the hormone auxin is in the formation of flowers at the periphery of the reproductive shoot apex. Mutants in regulators of polar auxin transport or in the auxin-responsive transcription factor MONOPTEROS (MP) form naked inflorescence "pins" lacking flowers. How auxin maxima and MP direct initiation of flower primordia is poorly understood. Here, we identify three genes whose expression is directly induced by auxin-activated MP that furthermore jointly regulate flower primordium initiation. These three genes encode known regulators of flower development: LEAFY (LFY), which specifies floral fate, and two AINTEGUMENTA-LIKE/PLETHORA transcription factors, key regulators of floral growth. Our study thus reveals a mechanistic link between flower primordium initiation and subsequent steps in flower morphogenesis. Finally, we uncover direct positive feedback from LFY to the auxin pathway. The auxin LFY module we describe may have been recruited during evolution to pattern other plant organ systems.

Control of Arabidopsis root development.

The Arabidopsis root has been the subject of intense research over the past decades. This research has led to significantly improved understanding of the molecular mechanisms underlying root development. Key insights into the specification of individual cell types, cell patterning, growth and differentiation, branching of the primary root, and responses of the root to the environment have been achieved. Transcription factors and plant hormones play key regulatory roles. Recently, mechanisms involving protein movement and the oscillation of gene expression have also been uncovered. Root gene regulatory networks controlling root development have been reconstructed from genome-wide profiling experiments, revealing novel molecular connections and models. Future refinement of these models will lead to a more complete description of the complex molecular interactions that give rise to a simple growing root.

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.

The LEAFY target LMI1 is a meristem identity regulator and acts together with LEAFY to regulate expression of CAULIFLOWER.

The timing of the switch from vegetative to reproductive development is crucial for species survival. The plant-specific transcription factor and meristem identity regulator LEAFY (LFY) controls this switch in Arabidopsis, in part via the direct activation of two other meristem identity genes, APETALA1 (AP1) and CAULIFLOWER (CAL). We recently identified five new direct LFY targets as candidates for the missing meristem identity regulators that act downstream of LFY. Here, we demonstrate that one of these, the class I homeodomain leucine-zipper transcription factor LMI1, is a meristem identity regulator. LMI1 acts together with LFY to activate CAL expression. The interaction between LFY, LMI1 and CAL resembles a feed-forward loop transcriptional network motif. LMI1 has additional LFY-independent roles in the formation of simple serrated leaves and in the suppression of bract formation. The temporal and spatial expression of LMI1 supports a role in meristem identity and leaf/bract morphogenesis.