Two modes of gene regulation by TFL1 mediate its dual function in flowering time and shoot determinacy of Arabidopsis.

Plant organ primordia develop successively at the shoot apical meristem (SAM). In Arabidopsis, primordia formed early in development differentiate into vegetative leaves, whereas those formed later generate inflorescence branches and flowers. TERMINAL FLOWER 1 (TFL1), a negative regulator of transcription, acts in the SAM to delay flowering and to maintain inflorescence meristem indeterminacy. We used confocal microscopy, time-resolved transcript profiling and reverse genetics to elucidate this dual role of TFL1. We found that TFL1 accumulates dynamically in the SAM reflecting its dual function. Moreover, TFL1 represses two major sets of genes. One set includes genes that promote flowering, expression of which increases earlier in tfl1 mutants. The other set is spatially misexpressed in tfl1 inflorescence meristems. The misexpression of these two gene sets in tfl1 mutants depends upon FD transcription factor, with which TFL1 interacts. Furthermore, the MADS-box gene SEPALLATA 4, which is upregulated in tfl1, contributes both to the floral transition and shoot determinacy defects of tfl1 mutants. Thus, we delineate the dual function of TFL1 in shoot development in terms of its dynamic spatial distribution and different modes of gene repression.

MicroRNA172 controls inflorescence meristem size through regulation of APETALA2 in Arabidopsis.

The APETALA2 (AP2) transcription factor regulates flower development, floral transition and shoot apical meristem (SAM) maintenance in Arabidopsis. AP2 is also regulated at the post-transcriptional level by microRNA172 (miR172), but the contribution of this to SAM maintenance is poorly understood. We generated transgenic plants carrying a form of AP2 that is resistant to miR172 (rAP2) or carrying a wild-type AP2 susceptible to miR172. Phenotypic and genetic analyses were performed on these lines and mir172 mutants to study the role of AP2 regulation by miR172 on meristem size and the rate of flower production. We found that rAP2 enlarges the inflorescence meristem by increasing cell size and cell number. Misexpression of rAP2 from heterologous promoters showed that AP2 acts in the central zone (CZ) and organizing center (OC) to increase SAM size. Furthermore, we found that AP2 is negatively regulated by AUXIN RESPONSE FACTOR 3 (ARF3). However, genetic analyses indicated that ARF3 also influences SAM size and flower production rate independently of AP2. The study identifies miR172/AP2 as a regulatory module controlling inflorescence meristem size and suggests that transcriptional regulation of AP2 by ARF3 fine-tunes SAM size determination.

Systematic analyses of the MIR172 family members of Arabidopsis define their distinct roles in regulation of APETALA2 during floral transition.

MicroRNAs (miRNAs) play important roles in regulating flowering and reproduction of angiosperms. Mature miRNAs are encoded by multiple MIRNA genes that can differ in their spatiotemporal activities and their contributions to gene regulatory networks, but the functions of individual MIRNA genes are poorly defined. We functionally analyzed the activity of all 5 Arabidopsis thaliana MIR172 genes, which encode miR172 and promote the floral transition by inhibiting the accumulation of APETALA2 (AP2) and APETALA2-LIKE (AP2-LIKE) transcription factors (TFs). Through genome editing and detailed confocal microscopy, we show that the activity of miR172 at the shoot apex is encoded by 3 MIR172 genes, is critical for floral transition of the shoot meristem under noninductive photoperiods, and reduces accumulation of AP2 and TARGET OF EAT2 (TOE2), an AP2-LIKE TF, at the shoot meristem. Utilizing the genetic resources generated here, we show that the promotion of flowering by miR172 is enhanced by the MADS-domain TF FRUITFULL, which may facilitate long-term silencing of AP2-LIKE transcription, and that their activities are partially coordinated by the TF SQUAMOSA PROMOTER-BINDING-LIKE PROTEIN 15. Thus, we present a genetic framework for the depletion of AP2 and AP2-LIKE TFs at the shoot apex during floral transition and demonstrate that this plays a central role in floral induction.

Functional Divergence of the Arabidopsis Florigen-Interacting bZIP Transcription Factors FD and FDP.

Flowering of many plant species depends on interactions between basic leucine zipper (bZIP) transcription factors and systemically transported florigen proteins. Members of the genus Arabidopsis contain two of these bZIPs, FD and FDP, which we show have largely complementary expression patterns in shoot apices before and during flowering. CRISPR-Cas9-induced null mutants for FDP flower slightly earlier than wild-type, whereas fd mutants are late flowering. Identical G-box sequences are enriched at FD and FDP binding sites, but only FD binds to genes involved in flowering and only fd alters their transcription. However, both proteins bind to genes involved in responses to the phytohormone abscisic acid (ABA), which controls developmental and stress responses. Many of these genes are differentially expressed in both fd and fdp mutant seedlings, which also show reduced ABA sensitivity. Thus, florigen-interacting bZIPs have distinct functions in flowering dependent on their expression patterns and, at earlier stages in development, play common roles in phytohormone signaling.

Gibberellins Act Downstream of Arabis PERPETUAL FLOWERING1 to Accelerate Floral Induction during Vernalization.

Regulation of flowering by endogenous and environmental signals ensures that reproduction occurs under optimal conditions to maximize reproductive success. Involvement of the growth regulator gibberellin (GA) in the control of flowering by environmental cues varies among species. Arabis alpina Pajares, a model perennial member of the Brassicaceae, only undergoes floral induction during vernalization, allowing definition of the role of GA specifically in this process. The transcription factor PERPETUAL FLOWERING1 (PEP1) represses flowering until its mRNA levels are reduced during vernalization. Genome-wide analyses of PEP1 targets identified genes involved in GA metabolism and signaling, and many of the binding sites in these genes were specific to the A. alpina lineage. Here, we show that the pep1 mutant exhibits an elongated-stem phenotype, similar to that caused by treatment with exogenous GA, consistent with PEP1 repressing GA responses. Moreover, in comparison with the wild type, the pep1 mutant contains higher GA4 levels and is more sensitive to GA prior to vernalization. Upon exposure to cold temperatures, GA levels fall to low levels in the pep1 mutant and in wild-type plants, but GA still promotes floral induction and the transcription of floral meristem identity genes during vernalization. Reducing GA levels strongly impairs flowering and inflorescence development in response to short vernalization treatments, but longer treatments overcome the requirement for GA. Thus, GA accelerates the floral transition during vernalization in A. alpina, the down-regulation of PEP1 likely increases GA sensitivity, and GA responses contribute to determining the length of vernalization required for flowering and reproduction.

Floral regulators FLC and SOC1 directly regulate expression of the B3-type transcription factor TARGET OF FLC AND SVP 1 at the Arabidopsis shoot apex via antagonistic chromatin modifications.

Integration of environmental and endogenous cues at plant shoot meristems determines the timing of flowering and reproductive development. The MADS box transcription factor FLOWERING LOCUS C (FLC) of Arabidopsis thaliana is an important repressor of floral transition, which blocks flowering until plants are exposed to winter cold. However, the target genes of FLC have not been thoroughly described, and our understanding of the mechanisms by which FLC represses transcription of these targets and how this repression is overcome during floral transition is still fragmentary. Here, we identify and characterize TARGET OF FLC AND SVP1 (TFS1), a novel target gene of FLC and its interacting protein SHORT VEGETATIVE PHASE (SVP). TFS1 encodes a B3-type transcription factor, and we show that tfs1 mutants are later flowering than wild-type, particularly under short days. FLC and SVP repress TFS1 transcription leading to deposition of trimethylation of Iysine 27 of histone 3 (H3K27me3) by the Polycomb Repressive Complex 2 at the TFS1 locus. During floral transition, after downregulation of FLC by cold, TFS1 transcription is promoted by SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), a MADS box protein encoded by another target of FLC/SVP. SOC1 opposes PRC function at TFS1 through recruitment of the histone demethylase RELATIVE OF EARLY FLOWERING 6 (REF6) and the SWI/SNF chromatin remodeler ATPase BRAHMA (BRM). This recruitment of BRM is also strictly required for SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9) binding at TFS1 to coordinate RNAPII recruitment through the Mediator complex. Thus, we show that antagonistic chromatin modifications mediated by different MADS box transcription factor complexes play a crucial role in defining the temporal and spatial patterns of transcription of genes within a network of interactions downstream of FLC/SVP during floral transition.

A regulatory circuit conferring varied flowering response to cold in annual and perennial plants.

The reproductive strategies of plants are highly variable. Short-lived annuals flower abundantly soon after germination, whereas longer-lived perennials postpone and spatially restrict flowering. We used CRISPR/Cas9 and interspecies gene transfer to understand divergence in reproductive patterns between annual and perennial crucifers. We show that in perennial Arabis alpina, flowering in response to winter cold depends on the floral integrator SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 15 (SPL15), whose activity is limited to older shoots and branches during cold exposure. In annuals, this regulatory system is conserved, but cold-induced flowering occurs in young shoots, without requirement for SPL15, through the photoperiodic pathway when plants return to warm. By reconstructing the annual response in perennials, we conclude that characteristic patterns of reproduction in annuals and perennials are conferred through variation in dependency on distinct flowering pathways acting in parallel.

Multi-layered Regulation of SPL15 and Cooperation with SOC1 Integrate Endogenous Flowering Pathways at the Arabidopsis Shoot Meristem.

Flowering is initiated in response to environmental and internal cues that are integrated at the shoot apical meristem (SAM). We show that SPL15 coordinates the basal floral promotion pathways required for flowering of Arabidopsis in non-inductive environments. SPL15 directly activates transcription of the floral regulators FUL and miR172b in the SAM during floral induction, whereas its paralog SPL9 is expressed later on the flanks of the SAM. The capacity of SPL15 to promote flowering is regulated by age through miR156, which targets SPL15 mRNA, and gibberellin (GA), which releases SPL15 from DELLAs. Furthermore, SPL15 and the MADS-box protein SOC1 cooperate to promote transcription of their target genes. SPL15 recruits RNAPII and MED18, a Mediator complex component, in a GA-dependent manner, while SOC1 facilitates active chromatin formation with the histone demethylase REF6. Thus, we present a molecular basis for assimilation of flowering signals and transcriptional control at the SAM during flowering.

Sample Preparation of Arabidopsis thaliana Shoot Apices for Expression Studies of Photoperiod-Induced Genes.

Plants produce new organs from a population of pluripotent cells which are located in specific tissues called meristems. One of these meristems, the shoot apical meristem (SAM), gives rise to leaves during the vegetative phase and flowers during the reproductive phase. The transition from vegetative SAM to an inflorescence meristem (IM) is a dramatic developmental switch, which has been particularly well studied in the model species Arabidopsis thaliana. This developmental switch is controlled by multiple environmental signals such as day length (or photoperiod), and it is accompanied by changes in expression of hundreds of genes. A major interest in plant biology is to identify and characterize those genes which are regulated in the stem cells of the SAM in response to the photoperiodic signals. In this sense, techniques such as RNA in situ hybridization (RNA ISH) have been very successfully employed to detect the temporal and spatial expression patterns of genes in the SAM. This method can be specifically optimized for photoperiodic-flowering studies. In this chapter, we describe improved methods to generate plant material and histological samples to be combined with RNA ISH in flowering-related studies.

Analysis of the Arabidopsis shoot meristem transcriptome during floral transition identifies distinct regulatory patterns and a leucine-rich repeat protein that promotes flowering.

Flowering of Arabidopsis thaliana is induced by exposure to long days (LDs). During this process, the shoot apical meristem is converted to an inflorescence meristem that forms flowers, and this transition is maintained even if plants are returned to short days (SDs). We show that exposure to five LDs is sufficient to commit the meristem of SD-grown plants to flower as if they were exposed to continuous LDs. The MADS box proteins SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) and FRUITFULL (FUL) play essential roles in this commitment process and in the induction of flowering downstream of the transmissible FLOWERING LOCUS T (FT) signal. We exploited laser microdissection and Solexa sequencing to identify 202 genes whose transcripts increase in the meristem during floral commitment. Expression of six of these transcripts was tested in different mutants, allowing them to be assigned to FT-dependent or FT-independent pathways. Most, but not all, of those dependent on FT and its paralog TWIN SISTER OF FT (TSF) also relied on SOC1 and FUL. However, this dependency on FT and TSF or SOC1 and FUL was often bypassed in the presence of the short vegetative phase mutation. FLOR1, which encodes a leucine-rich repeat protein, was induced in the early inflorescence meristem, and flor1 mutations delayed flowering. Our data contribute to the definition of LD-dependent pathways downstream and in parallel to FT.

Aa TFL1 confers an age-dependent response to vernalization in perennial Arabis alpina.

Flowering of many plants is induced by environmental signals, but these responses can depend on the age of the plant. Exposure of Arabidopsis thaliana to vernalization (winter temperatures) at germination induces flowering, whereas a close perennial relative Arabis alpina only responds if exposed when at least 5 weeks old. We show that vernalization of these older A. alpina plants reduces expression of the floral repressor PEP1 and activates the orthologs of the Arabidopsis flowering genes SOC1 (Aa SOC1) and LFY (Aa LFY). By contrast, when younger plants are vernalized, PEP1 and Aa SOC1 mRNA levels change as in older plants, but Aa LFY is not expressed. We demonstrate that A. alpina TFL1 (Aa TFL1) blocks flowering and prevents Aa LFY expression when young plants are exposed to vernalization. In addition, in older plants, Aa TFL1 increases the duration of vernalization required for Aa LFY expression and flowering. Aa TFL1 has similar functions in axillary shoots, thus ensuring that following a flowering episode vegetative branches are maintained to continue the perennial life cycle. We propose that Aa TFL1 blocks flowering of young plants exposed to vernalization by setting a threshold for a flowering pathway that is increased in activity as the shoot ages, thus contributing to several perennial traits.

PEP1 regulates perennial flowering in Arabis alpina.

Annual plants complete their life cycle in one year and initiate flowering only once, whereas perennials live for many years and flower repeatedly. How perennials undergo repeated cycles of vegetative growth and flowering that are synchronized to the changing seasons has not been extensively studied. Flowering is best understood in annual Arabidopsis thaliana, but many closely related species, such as Arabis alpina, are perennials. We identified the A. alpina mutant perpetual flowering 1 (pep1), and showed that PEP1 contributes to three perennial traits. It limits the duration of flowering, facilitating a return to vegetative development, prevents some branches from undergoing the floral transition allowing polycarpic growth habit, and confers a flowering response to winter temperatures that restricts flowering to spring. Here we show that PEP1 is the orthologue of the A. thaliana gene FLOWERING LOCUS C (FLC). The FLC transcription factor inhibits flowering until A. thaliana is exposed to winter temperatures, which trigger chromatin modifications that stably repress FLC transcription. In contrast, PEP1 is only transiently repressed by low temperatures, causing repeated seasonal cycles of repression and activation of PEP1 transcription that allow it to carry out functions characteristic of the cyclical life history of perennials. The patterns of chromatin modifications at FLC and PEP1 differ correlating with their distinct expression patterns. Thus we describe a critical mechanism by which flowering regulation differs between related perennial and annual species, and propose that differences in chromatin regulation contribute to this variation.

FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis.

In plants, seasonal changes in day length are perceived in leaves, which initiate long-distance signaling that induces flowering at the shoot apex. The identity of the long-distance signal has yet to be determined. In Arabidopsis, activation of FLOWERING LOCUS T (FT) transcription in leaf vascular tissue (phloem) induces flowering. We found that FT messenger RNA is required only transiently in the leaf. In addition, FT fusion proteins expressed specifically in phloem cells move to the apex and move long distances between grafted plants. Finally, we provide evidence that FT does not activate an intermediate messenger in leaves. We conclude that FT protein acts as a long-distance signal that induces Arabidopsis flowering.

The transcription factor FLC confers a flowering response to vernalization by repressing meristem competence and systemic signaling in Arabidopsis.

Floral development at the Arabidopsis shoot apical meristem occurs in response to environmental cues that are perceived in different tissues. Photoperiod is detected in the vascular tissue of the leaf (phloem) and promotes production of a systemic signal that induces flowering at the meristem. Vernalization, the response to winter temperatures, overcomes a block on photoperiodic floral induction. In Arabidopsis, this block is caused by inhibitors of flowering that comprise several related MADS-box transcription factors, the most prominent of which is FLC. We show that FLC delays flowering by repressing production in the leaf of at least two systemic signals, one of which is controlled by the RAF kinase inhibitor-like protein FT. Reducing expression of these signals indirectly represses expression of genes involved in floral induction at the meristem. In addition, FLC expression in the meristem impairs response to the FT signal by directly repressing expression of the SOC1 MADS-box transcription factor and preventing up-regulation of the bZIP transcription factor FD. Repression of genes acting at multiple levels in this hierarchy is required for the extreme delay in flowering caused by FLC. An FLC:HA fusion protein binds directly in vivo to the promoter regions of FD and SOC1 and to the first intron of FT. Thus vernalization relieves transcriptional repression of key regulatory genes in both the leaf and meristem, allowing production of systemic signals in the leaves and conferring competence on the meristem to respond to these signals.

CONSTANS acts in the phloem to regulate a systemic signal that induces photoperiodic flowering of Arabidopsis.

Flower development at the shoot apex is initiated in response to environmental cues. Day length is one of the most important of these and is perceived in the leaves. A systemic signal, called the floral stimulus or florigen, is then transmitted from the leaves through the phloem and induces floral development at the shoot apex. Genetic analysis in Arabidopsis identified a pathway of genes required for the initiation of flowering in response to day length. The nuclear zinc-finger protein CONSTANS (CO) plays a central role in this pathway, and in response to long days activates the transcription of FT, which encodes a RAF-kinase-inhibitor-like protein. We show using grafting approaches that CO acts non-cell autonomously to trigger flowering. Although CO is expressed widely, its misexpression from phloem-specific promoters, but not from meristem-specific promoters, is sufficient to induce early flowering and complement the co mutation. The mechanism by which CO triggers flowering from the phloem involves the cell-autonomous activation of FT expression. Genetic approaches indicate that CO activates flowering through both FT-dependent and FT-independent processes, whereas FT acts both in the phloem and the meristem to trigger flowering. We propose that, partly through the activation of FT, CO regulates the synthesis or transport of a systemic flowering signal, thereby positioning this signal within the established hierarchy of regulatory proteins that controls flowering.

Interactions between gene activity and cell layers during floral development.

The DEFICIENS (DEF) gene is required for establishing petal and stamen identity in Antirrhinum and is expressed in all three layers of the floral meristem in whorls 2 and 3. Expression of DEF in a subset of meristem layers gives rise to organs with characteristic shapes and cell types, reflecting altered patterns and levels of DEF gene activity. To determine how the contributions of layers and gene activity interact, we exploited a DEF allele which carries a transposon insertion in the MADS box region to generate periclinal chimeras expressing alleles with different activities. By comparing the phenotype, development and expression patterns of these chimeras we show that expression of DEF in L1 makes a major contribution to morphology in whorl 2, irrespective of the allele. By contrast L1 expression is largely unable to rescue whorl 3, possibly because of a non-autonomous inhibitor of DEF activity in this whorl.