What a tangled web it weaves: auxin coordination of stem cell maintenance and flower production

Abstract

Robust agricultural yields require consistent flower production throughout fluctuating environmental conditions. Floral primordia are produced in the inflorescence meristem, which contains a pool of continuously dividing stem cells. Daughter cells of these divisions either retain stem cell identity or are pushed to the SAM periphery, where they become competent to develop into floral primordia after receiving the appropriate signal. Thus, flower production is inherently linked to regulation of the stem cell pool. The plant hormone auxin promotes flower development throughout its early phases and has been shown to interact with the molecular pathways regulating stem cell maintenance. Here, we will summarize how auxin signaling contributes to stem cell maintenance and promotes flower development through the early phases of initiation, outgrowth, and floral fate establishment. Recent advances in this area suggest that auxin may serve as a signal that integrates stem cell maintenance and new flower production.

A review summarizing recent findings of how auxin function regulates the molecular pathways controlling stem cell maintenance and early flower development in Arabidopsis.

Introduction

Robust flower production underlies the reproductive success of plants and, as it is inherently linked to agricultural yields, has been associated with several domesticated traits across species. Constant stem cell division in the shoot apical meristem (SAM) is critical to ensuring continuous lateral organ production, which encompasses leaf production during vegetative development and production of axillary meristems and flowers during the reproductive phase (Wang and Jiao, 2018). As the stem cells divide, the new daughter cells produced either remain stem cells in the center of the SAM (the central zone) or are pushed to the outer edges (the peripheral zone) where they become competent to develop into new organs (Fig. 1A) (Somssich et al., 2016; Soyars et al., 2016; Willoughby and Nimchuk, 2021). This balance between stem cell proliferation and new flower production must be well-coordinated to prevent stem cell exhaustion and consequent cessation of flower production.

Fig. 1.

Overview of SAM organization and auxin dynamics. (A) Organization of the shoot apical meristem (SAM.) (B) Auxin dynamics in the SAM. The transcriptional domains of TAA and YUCCAs are noted. The central zone is indicated with a dashed circle. The two auxin transport steps we propose are required for primordium initiation are labeled with arrows.

Plant hormones integrate endogenous and environmental signals into changes in developmental programs throughout the plant. In the SAM, hormones are involved in both stem cell proliferation and new lateral organ production. In particular, the hormone auxin is essential for lateral organ development, as it specifies the position of new lateral organs at the SAM periphery (Wang and Jiao, 2018; Lopes et al., 2021). In this review, we will explore how auxin signaling integrates two interconnected developmental systems: those maintaining the stem cell pool by promoting stem cell identity and those initiating and promoting floral development in the SAM periphery. As the molecular components of these pathways are detailed extensively in Arabidopsis, we will focus on integrating recent advances in this field into a comprehensive and up-to-date model and highlighting promising areas for future study. Many reviews have compared stem cell and flower development across species, and we refer interested readers to these (Pautler et al., 2013; Galli and Gallavotti, 2016; Nardmann et al., 2016; Fletcher, 2018; Kitagawa and Jackson, 2019; Eshed Williams, 2021). In the first section, we will provide a brief overview of auxin biology. Then, we will discuss the interplay of auxin and stem cell signaling in the inflorescence meristem. Finally, we will discuss the role of auxin in the earliest phases of flower development. Key molecular components at the junction of auxin function, stem cell regulation, and early flower development are shown in Table 1.

Table 1.

Key components in auxin regulation of stem cells and flower production

Biological role	Gene name	Functional description
Stem cell signaling	CLV1	CLE receptor, represses WUS expression
	CLV2/CRN	Receptor/pseudokinase pair, regulates flower outgrowth
	CLV3	CLE ligand in stem cells
	WUS	Represses differentiation and promotes stem cell division
	STM	Promotes meristematic identity
Auxin response factors	ARF3 (ETT)	Repressor ARF (class B), auxin receptor
	ARF5 (MP)	Activator (class A) ARF
Auxin transport	PIN1	Auxin efflux carrier
Auxin biosynthesis	TAA	Catalyses first step in auxin biosynthesis
	YUC	Catalyses rate-limiting auxin biosynthesis step
Floral regulators	LFY	Promotes floral identity, regulates initiation and outgrowth together with auxin transport
	ANT/AIL6	Promote floral identity, regulates initiation and outgrowth together with auxin transport
	DRN/DRNL/PUCHI	Promote floral identity by LFY activation, involved in initiation by activating ANT/AIL6 with ARF5
	SVP/AGL24/SOC1	Promote inflorescence identity early in floral development and floral identity through LFY activation

Overview of auxin biology

While several auxin biosynthesis pathways have been described, the predominant one involves conversion of tryptophan to indole-3-pyruvic acid (IPyA) by the TRYPTOPHAN AMINOTRANSFERASE (TAA) family enzymes. IPyA is then converted by the YUCCA (YUC) family enzymes to indole-3-acetic acid (IAA), the most abundant active auxin found in plants. The YUCCA-catalysed conversion is often the rate limiting step in auxin biosynthesis (Mashiguchi et al., 2011; Won et al., 2011). Environmental cues often activate auxin biosynthesis via the TAA/YUC pathway to modulate plant development, indicating that this pathway is one of the most physiologically relevant (Franklin et al., 2011; Sun et al., 2012; Bellstaedt et al., 2019; van der Woude et al., 2019). Following its biosynthesis, auxin is transported to distant tissues via the PIN-FORMED (PIN) family of auxin efflux carriers and certain subclasses of ATP-binding cassette (ABC) transporters (Geisler et al., 2017; Sauer and Kleine-Vehn, 2019). The PINs have the most well-described roles in plant development, regulating diverse developmental processes such as apical–basal axis establishment in embryogenesis, tropic growth responses, and organogenesis (Reinhardt et al., 2000; Jenik et al., 2007; Adamowski and Friml, 2015; Han et al., 2021). Additionally, two other protein families are involved in more local auxin movement: AUXIN RESISTANT (AUX1/LAX) proteins are auxin influx carriers, and the PIN-LIKES (PILs) are involved in intracellular auxin sequestration (Singh et al., 2018; Sauer and Kleine-Vehn, 2019). Finally, several recent reports have proposed that auxin travels through plasmodesmata to regulate various developmental processes (Gao et al., 2020; Mellor et al., 2020; Mehra et al., 2022).

In responding cells, auxin influences plant growth and development in one of two ways: first, through activation of rapid responses, such as cellular elongation via cell wall acidification or rapid alterations in the proteome; and second, by activation of transcriptional programs that initiate developmental changes (Ang and Østergaard, 2023). Different auxin perception pathways can feed into rapid or long-term responses through their interaction with transcriptional or cellular signaling machinery (Ang and Østergaard, 2023; Pérez-Henríquez and Yang, 2023). An auxin perception pathway via AUXIN BINDING PROTEIN (ABP1) and recently identified homologs ABL1 and ABL2 has been hypothesized to facilitate rapid cellular auxin responses (Gelová et al., 2021; Yu et al., 2022, Preprint; Friml et al., 2022). Currently, there are no data revealing a role for this pathway in auxin regulation of stem cells or flower production, so we will focus on how transcriptional responses to auxin alter SAM physiology and flower production.

Transcriptional responses to auxin typically occur via the classical auxin perception and signal transduction pathway. TRANSPORT INHIBITOR RESPONSE 1 (TIR1)/AUXIN SIGNALING F-BOX (AFB) F box proteins, which are components of an SCF-type ubiquitin ligase, bind to auxin and initiate the degradation of INDOLE-3-ACETIC ACID INDUCIBLE (IAAs), which repress transcription of auxin responsive genes. IAAs also interact with and repress class A auxin response factors (ARFs), which activate the expression of auxin responsive genes. Thus, upon auxin perception via TIR1/AFB, IAAs are degraded, releasing ARFs to activate new developmental programs through transcriptional responses to auxin signaling (Salehin et al., 2015; Leyser, 2018; Ang and Østergaard, 2023). Though TIR1/AFB-mediated auxin perception primarily targets long-term auxin transcriptional responses, it has also been shown to contribute to some rapid auxin-induced cellular elongation processes (Fendrych et al., 2016, 2018).

As ARFs are the direct executors of auxin-induced transcriptional changes, endogenous and environmental regulation of ARF function represents a major hub at which auxin signals are integrated into developmental outcomes. ARFs are divided into three phylogenetic groups: class A (activators) and classes B and C (repressors). While the precise mechanisms of class B and C ARF transcriptional regulation remain elusive, class A ARFs have been more thoroughly described. In the absence of auxin, they act as repressors of auxin responsive genes, but upon IAA degradation following auxin perception, they behave as activators (Roosjen et al., 2018; Cancé et al., 2022). Additionally, class A ARFs can form cytoplasmic liquid droplets at higher concentrations, sequestering them from nuclear accumulation and transcriptional activity and thus reducing the transcriptional response to auxin. ARF liquid-droplet formation fluctuates with developmental and environmental context, suggesting that its biophysical state may serve as a nexus of environmental regulation of auxin response (Powers et al., 2019; Jing et al., 2022). The most relevant ARFs to stem cell regulation and early flower development, which will be discussed in the following sections, are ARF3 (class B) and ARF5 (class A). Intriguingly, several reports suggest that ARF3 may be an auxin receptor, as it directly binds auxin and initiates transcriptional responses following this auxin perception (Simonini et al., 2018; Kuhn et al., 2020).

The role of auxin in stem cell maintenance

Overview of stem cell signaling and auxin dynamics in the SAM

Stem cells must maintain their pluripotent identity and be continuously renewed to ensure continual lateral organ production throughout the life of the plant. The family of CLAVATA receptors and their peptide ligands, the CLAVATA3/EMBRYO-SURROUNDING REGIONs (CLEs), have been shown to repress stem cell proliferation in Arabidopsis. This occurs, at least partly, through transcriptional repression of WUSCHEL (WUS), which encodes a transcription factor that represses differentiation and promotes stem cell identity. WUS is expressed in a group of cells in the center of the SAM called the organizing center (OC). The OC sits directly below the stem cells in the central zone (CZ). WUS travels through plasmodesmata to the stem cells, where it induces expression of CLV3, which encodes a CLE peptide. Mature processed CLV3 peptide (CLV3p) then diffuses back down to the OC where it is perceived by the receptor-like kinase CLV1, causing it to be internalized. Through an incompletely understood signaling cascade, this perception of CLV3p by CLV1 leads to dampening of WUS expression, establishing a negative feedback loop that maintains the size of the SAM (Soyars et al., 2016; Willoughby and Nimchuk, 2021).

Additionally, the transcription factor SHOOTMERISTEMLESS (STM) is necessary for establishment and maintenance of the SAM. Strong stm mutants do not establish a SAM during embryonic development (Long et al., 1996). Early work implied that STM and WUS regulated stem cell identity independently of each other, because overexpression of one could not compensate for loss of the other, and because loss of either resulted in failure to establish (in the case of stm) or maintain (in the case of wus) a SAM (Mayer et al., 1998; Lenhard et al., 2002). Recent evidence suggests that STM interacts with WUS to maintain normal CLV3 expression in stem cells, and that WUS activates STM expression, while STM does not activate WUS (Su et al., 2020). STM, like WUS, is mobile in the SAM, and its intercellular trafficking is required to maintain the SAM and promote proper axillary meristem formation and organ boundary establishment (Balkunde et al., 2017; Kitagawa et al., 2022). STM is found throughout the SAM, except in incipient floral primordia, where its transcriptional down-regulation is a hallmark of floral specification (Heisler et al., 2005; Scofield et al., 2018; Chung et al., 2019).

To understand how auxin dynamics influence stem cell regulation, it is important to first understand the spatial dynamics of auxin biosynthesis, transport, perception, and response in the SAM. Auxin researchers have used synthetic fluorescent reporters to assess auxin perception and response localization throughout the SAM. Auxin perception can be detected using the DII reporter, which contains the degron domain of IAAs. Thus, a fluorescent protein tagged with the DII domain will be degraded in cells that are perceiving auxin (Brunoud et al., 2012). Multiple groups have examined the distribution of DII signal in the SAM and found that an auxin perception maximum occurs at P0, the first detectable incipient primordium that begins bulging away from the SAM (Brunoud et al., 2012; Liao et al., 2015; Galvan-Ampudia et al., 2020). This is consistent with the requirement for localized auxin increases to initiate a flower. Additionally, auxin is perceived at moderate levels in the central zone, where stem cells are located. The areas of lowest auxin perception are in the boundary regions between stem cells and incipient floral primordia and between adjacent primordia. Similarly, auxin-induced gene expression, which can be detected using a synthetic promoter containing Auxin Response Elements (AuxREs) (DR5), are the strongest in the incipient primordia at the periphery of the SAM, lower in the central zone, and not detected in the boundary regions (Ma et al., 2019; Galvan-Ampudia et al., 2020).

Most auxin biosynthesis in the SAM likely occurs in the incipient lateral organs via the TAA/YUC pathway. While TAA enzymes are expressed in both the central zone and incipient primordia, the YUCs, which represent the final rate-limiting step in the major biosynthesis pathway, are primarily expressed in incipient floral primordia or the boundary between primordia and the peripheral zone (Mashiguchi et al., 2011; Yadav et al., 2020, Preprint). Only very low and patchy YUC expression, if any, is detected in the central zone (Galvan-Ampudia et al., 2020). Altogether, these data imply that auxin is likely to be primarily synthesized in the primordia and that the auxin sensing and transcriptional responses that occur in the center of the SAM could be initiated by auxin transported into the central zone from the primordia (Fig. 1B).

Evidence from a few recent reports provides further support for this hypothesis. First, careful analysis of PIN1 polarity in the SAM showed that auxin flux via PIN1 supplies auxin from P3 to P5 to the center of the SAM (Heisler et al., 2005; Galvan-Ampudia et al., 2020). This shows that auxin is transported into the SAM from the floral primordia before they separate from the SAM later in floral development. Additionally, another study showed that removing auxin from the SAM pharmacologically or genetically mimics the effects of removal of primordia. For instance, the yuc1/2/4/6 quadruple mutant has an enlarged SAM, indicating that auxin negatively regulates SAM size. Furthermore, applying exogenous auxin reduces the size of the SAM, shrinking the stem cell pool specifically; conversely, blocking auxin perception with the TIR1/AFB inhibitor auxinole increases SAM size (Shi et al., 2018). If floral primordia are a primary source of auxin for the SAM, then removal of floral primordia should result in SAM expansion. This is in fact the case; removal of all floral primordia at the P2 stage and older caused a reduction in auxin signaling across the SAM and an increase in the size of the stem cell pool (Shi et al., 2018). These data support the hypothesis that most auxin biosynthesis in the SAM occurs in incipient primordia. Additionally, these data suggest that auxin restricts the size of the stem cell pool.

Auxin as a positive regulator of the stem cell pool

In contrast to the data discussed above, several lines of evidence paradoxically support the idea that continuous low levels of auxin are required to maintain the stem cell pool. First, stem cells also sense and respond transcriptionally to auxin, as discussed earlier. Additionally, expression of the stabilized mutant IAA protein bdl (IAA12) in the CLV3 domain initially causes an expansion of SAM size due to the reduction in auxin signaling, and its continued expression causes termination of the SAM, even in clv3 mutants that have an expanded SAM size (Ma et al., 2019).

Additionally, recent evidence has suggested that CLAVATA signaling promotes auxin function in the SAM to maintain the stem cell pool. First, transcriptional auxin responses were reduced in the clv3 mutant background, suggesting that CLV3p signaling maintains appropriate auxin levels in the SAM (Ma et al., 2019). Furthermore, a recent report describes a novel stem cell maintenance phenotype of clv1 null mutants that appears to be linked to auxin function. Null clv1 mutants show primary inflorescence termination (PIT), in which the primary SAM produces only a few flowers before ceasing flower production and internode elongation. clv1 SAMs that display PIT also show a reduction in auxin-induced gene expression (John et al., 2023). Curiously, clv1 plants grown in the heat, in which endogenous auxin levels are elevated due to the thermomorphogenic response (Delker et al., 2022), do not show the PIT phenotype, suggesting that elevating auxin levels can suppress the stem cell maintenance defects of clv1 mutants. Indeed, genetically removing auxin biosynthesis from the clv1 background, thus reducing the endogenous levels of auxin even further, enhances PIT penetrance at lower temperatures and reduces the ability of heat to suppress PIT. These data suggest that CLV1 promotes the constitutive low levels of auxin required for meristem maintenance. Intriguingly, this may be independent of WUS function, as the WUS expression domain remains unaltered in clv1 SAMs displaying PIT (John et al., 2023).

While genetically removing auxin biosynthesis from clv3 mutants causes an increase in SAM size, consistent with auxin’s role as a negative regulator of stem cell proliferation, clv3 yuc1/4 mutants show a reduction in stem fasciation (John et al., 2023). This suggests that stem cell proliferation and stem fasciation are separable, and that auxin positively regulates some stem cell functions while negatively regulating others. In clv1, yuc4 mutation also suppresses carpel numbers, which provide a quantitative measure for floral meristem size, indicating that auxin promotes floral stem cell proliferation (John et al., 2023). Overall, recent data suggest that the low, constitutive levels of auxin required to maintain the SAM are maintained by CLAVATA signaling.

Auxin regulation of key stem cell maintenance-related transcription factors

Auxin influences the function of several transcription factors known to regulate stem cell maintenance, including ARF3, WUS, and DRN/DRNL. The authors of a recent study propose that ARF3 travels from the peripheral zone to the organizing center to repress WUS expression there (Zhang et al., 2022). arf3 mutants have increased SAM sizes, and ARF3 that is targeted to the nucleus with an NLS tag, thus losing cell-to-cell mobility, cannot fully complement the stem cell defects of arf3 mutants. Furthermore, WUS protein levels are elevated in arf3 mutants complemented with the nuclear-targeted ARF3 as opposed to the native ARF3, indicating that ARF3’s mobility is required for its WUS repression (Zhang et al., 2022). As ARF3 is a possible auxin receptor, this could provide a direct link from auxin perception to repression of stem cell identity and a restriction of the stem cell pool size via WUS.

Next, there is evidence that WUS, which promotes stem cell identity through activation of CLV3 expression, negatively regulates auxin function in the SAM. First, one study using an inducible form of WUS identified genes involved in auxin biosynthesis as being down-regulated by WUS induction (Yadav et al., 2013). Additionally, depleting WUS from the OC using a nanobody system leads to an increase in auxin transcriptional responses in the stem cells. Furthermore, long-term WUS induction causes expansion of the stem cell region and a reduction in auxin transcriptional output (Ma et al., 2019). Since auxin depletion in the SAM was also shown to cause expansion, this is consistent with WUS repression of auxin transcriptional responses (Shi et al., 2018). Additionally, Ma et al suggest that WUS buffers auxin responses by allowing the moderate levels of auxin required to maintain the stem cell pool and preventing auxin levels from becoming too high. While the study provides convincing evidence that certain low levels of auxin are required to maintain the stem cell pool (the pCLV3:bdl phenotype discussed earlier), direct evidence showing WUS activates this low-level auxin responses remains to be elucidated. As clv1 mutants showing PIT and reduced auxin responses have an unaltered WUS domain, this suggests that the activation of low levels of auxin function in stem cells may be separable from WUS activity. Furthermore, in floral stem cells, increased WUS levels and genetic reductions in auxin biosynthesis in clv mutants have opposite effects on floral organ numbers (Laux et al., 1996; John et al., 2023). Lastly, a commonly used method to study WUS function in the SAM is to block WUS mobility through closing plasmodesmata (Daum et al., 2014; Ma et al., 2019). As it is now documented that other transcription factors that regulate stem cell function, including STM and ARF3, move through the SAM to exert their effects on stem cells, caution should be used when interpreting experiments that use this methodology.

The transcription factors DORNRÖSCHEN (DRN) and DORNRÖSCHEN-LIKE (DRNL), which promote auxin biosynthesis, also negatively regulate stem cell proliferation (Luo et al., 2018). One report shows that the drn drnl double mutant reverts the SAM termination phenotype of pCLV3:bdl plants, suggesting that DRN/DRNL function is responsible for SAM termination when auxin responses are repressed. Conversely, overexpression of DRN in the CLV3 domain caused SAM termination. Additionally, the SAM size of drn drnl mutants is increased, corresponding to a reduced CLV3 domain and, as a result, an increased WUS domain, consistent with the effects of reduced auxin on SAM size. Luo et al. (2018) suggest that DRN/DRNL are responsible for activation of CLV3 expression, though any regulation may be indirect and additive with additional unknown regulators as no DRN/DRNL binding to the CLV3 promoter is detected, and the drn drnl mutant does not fully recapitulate clv3 stem cell defects.

Additionally, this report shows that DRN expression in the SAM is negatively regulated by auxin. Mutating AuxREs in the DRN promoter causes an expansion of the DRN domain emanating from the stem cell pool. The authors suggest that ARF5 facilitates this repression by showing binding of ARF5 to the DRN promoter (Luo et al., 2018). Interestingly, ARF5, an activator ARF, activates DRN expression in the embryo (Cole et al., 2009; Cancé et al., 2022). Additionally, a recent report also showed that ARF5 can activate DRN in the peripheral zone in the drnl mutant background, suggesting that ARF5 regulation of DRN expression is context-dependent (Dai et al., 2023). One possibility for how ARF5 could act as a repressor in the SAM could be through interacting with IAA32/34, which can be phosphorylated and thus stabilized by Transmembrane Kinase 1 (TMK1) upon auxin perception by ABPs (Cao et al., 2019). Stabilized IAA32/34 could thus allow ARF5 to repress gene expression in certain tissues. Overall, these results suggest that auxin negatively regulates DRN, which in turn activates CLV3 thereby repressing WUS, ultimately restricting the size of the stem cell pool (Luo et al., 2018). As DRN/DRNL are known to activate auxin biosynthesis, and because the published transcriptional domain of DRNL is in incipient floral primordia, it may be possible that DRNL-activated auxin in the peripheral zone represses DRN via ARF5, and as DRN promotes stem cell identity through indirect regulation of CLV3 and WUS, this would cause auxin regulation of DRN to restrict the size of the stem cell pool (Chandler and Werr, 2011, 2017; Eklund et al., 2011). Exploring the roles of DRN/DRNL in auxin-mediated stem cell maintenance may elucidate the link between stem cell signaling and activation of auxin biosynthesis necessary for both stem cell maintenance and potentially for initiation of primordia.

Although STM down-regulation via auxin is a key step in initiation of floral primordia (Heisler et al., 2005; Chung et al., 2019), one report suggests that STM influences the expression of genes involved in auxin biosynthesis, transcriptional response, and transport in the SAM, suggesting a more complex interaction between STM and auxin (Scofield et al., 2018). As STM may activate some auxin responses in the SAM, it may be that STM activates the low levels of auxin required to maintain the SAM, perhaps in conjunction with CLAVATA signaling. Additionally, it would be worth investigating whether the STM–WUS interaction that promotes CLV3 expression also regulates the expression of auxin-related genes (Su et al., 2020). Alternatively, STM may participate in limiting the amount of auxin in the stem cells.

In summary, auxin is a negative regulator of SAM size (Fig. 2). Multiple groups and lines of evidence have shown that reducing auxin in the SAM causes an increase in the size of the stem cell pool. WUS antagonizes auxin function, acting as a signal to promote cell proliferation. Additionally, low levels of auxin are required to maintain the stem cell pool, as clv1 mutants and pCLV3:bdl plants show terminated SAMs. Exactly how CLAVATA signaling activates these low levels of auxin remains unclear.

Fig. 2.

Summary of the molecular pathways regulating stem cell maintenance and their effect on auxin function (noted as auxin). Indirect interactions are shown by dashed lines. An orthogonal view of the SAM is outlined, with the floral primordium highlighted by black shadow.

The role of auxin in flower production

Overview of flower production

The SAM transitions to producing flowers as lateral organs when it receives the signal to flower, FLOWERING TIME (FT), whose mRNA travels from the leaves to the SAM. FT initiates transcriptional programs that make the peripheral zone, and thus new incipient lateral organs, competent to develop into flowers instead of leaves or axillary meristems (Quiroz et al., 2021). The regulation of flowering time is highly complex and has been reviewed extensively elsewhere (Campos-Rivero et al., 2017; Freytes et al., 2021; Brightbill and Sung, 2022; Osnato et al., 2022). Curiously, no role for auxin regulation of the flowering time integrators FT and SUPPRESSOR OF OVEREXPRESSOR OF CO1 (SOC1) has been proposed in Arabidopsis. Auxin influences flowering through the activation of the third flowering time integrator, LEAFY (LFY), in the incipient organ, which will be subsequently discussed (Yamaguchi et al., 2014a).

The phases of flower development include initiation, outgrowth of primordia, floral fate establishment and reinforcement, meristem determinacy, and outgrowth of floral organs (Smyth et al., 1990). Flowers initiate at the SAM periphery when a local auxin maximum triggers the downstream floral transcriptional program (Galvan-Ampudia et al., 2020). In Arabidopsis, flowers initiate at 137.5° intervals. The positioning of flowers, called phyllotaxy, is regulated by hormonal signals (Reinhardt et al., 2003; Heisler et al., 2005). During initiation, floral fate establishment begins, as certain factors involved in initiation are also key factors in promoting and solidifying floral identity. Following initiation, an initial period of cell proliferation follows; the floral primordium forms a bulge that is clearly separable from the SAM. This morphology is described as stage 2 of flower development (Smyth et al., 1990). Concurrently, the auxin-induced floral fate transcriptional program will amplify its own expression, as well as that of additional floral identity components, thus further promoting floral fate in the new organ (Yamaguchi et al., 2013, 2016). Epigenetic modifications further reinforce this transcriptional program and thus floral fate (Campos-Rivero et al., 2017). Additionally, the floral fate transcriptional program will prevent reversion of the floral meristem to an inflorescence meristem by repressing inflorescence identity markers in the developing primordia (Gregis et al., 2009; Grandi et al., 2012). Finally, activation of transcription factors specific to flower development according to the ABCE floral organ identity model will allow development of specific floral organs, ultimately leading to the production of a mature flower (Goslin et al., 2023). The role of auxin in floral organ development and floral meristem determinacy has been reviewed elsewhere, and we will focus here on the first three phases of early floral development (Cucinotta et al., 2021).

Genetic evidence shows that flower initiation, outgrowth, and floral fate establishment are partially separable (Fig. 3). However, these processes cannot be dissected genetically in isolation, as several genes regulate multiple early developmental phases. For example, some genes that are involved in outgrowth and floral identity establishment also cooperate with auxin transport to promote initiation, as revealed by sensitized genetic backgrounds. This regulatory complexity reflects the fact that the division between the SAM and incipient primordia is hazy; during the initiation stage, the new primordia acquire a commitment to a new lineage while remaining in the SAM itself. During outgrowth, the separation of the primordia from the SAM increases as cell proliferation occurs, and by the time floral fate is established and reinforced, the primordia have disconnected even more from the SAM. In the following sections, we will discuss the role of auxin during these interconnected phases of initiation of primordia, outgrowth, and floral fate establishment. Known auxin-related pathways involved in the early phases of flower development are summarized in Fig. 4.

Fig. 3.

Early flower development mutant phenotypes. Illustrations of mutants showing defects in floral initiation (A), outgrowth (B), and floral fate establishment (C) are accompanied by orthogonal SAM outlines with the primordium highlighted in black shadow. Mature flowers are shown as lines with circles on the end, and branches are shown as lines with triangles.

Fig. 4.

Summary of the auxin-related molecular pathways during early flower development. (A) Auxin-dependent STM repression during initiation. (B) The CLAVATA signaling network promoting auxin function during initiation and outgrowth. (C) Auxin-regulated transcription factor network involved in the early phases of flower development, including initiation, outgrowth, floral fate establishment, and reinforcement of floral fate. Dashed lines show interactions that reinforce floral fate.

Initiation

The primary evidence for auxin’s role in initiation of floral primordia is the pin-like inflorescences of the arf5 and pin1 phenotypes. Auxin signals through ARF5, which is expressed in the peripheral zone of the SAM, to activate the transcriptional program required for initiation of floral primordia. While PIN1 is known to transport auxin to generate new auxin maxima and subsequent initiation of primordia, the source of this auxin is not clear. A recent report from Galvan-Ampudia et al. (2020) suggested that the auxin responsible for floral initiation comes from protrusions of the higher-auxin zone in the center of the SAM into the peripheral zone. However, as discussed above, the YUC expression domain suggests that the older organ primordia seem to be the primary source of auxin in the SAM (Galvan-Ampudia et al., 2020; Yadav et al., 2020, Preprint). These observations could suggest that continued formation of primordia might require two steps of auxin transport via PIN1: first, auxin export from older primordia to the central zone, and second, auxin transport from the central zone towards future primordium sites (Fig. 1B; Galvan-Ampudia et al., 2020). This model is consistent with the pin1 phenotype, because in the absence of PIN1, auxin could neither be imported to the SAM from incipient primordia, nor exported to the newest site of primordium initiation from the central zone. Additionally, PIN1 polarity supports this model, as it is polarized toward the central zone in P3–P5, while PIN1 in the central zone is polarized outward to the periphery (Heisler et al., 2005; Galvan-Ampudia et al., 2020). Finally, the observation that auxin moves through cells, as opposed to with cells as they divide, in the SAM provides additional support for this model, as it suggests that the auxin required for initiation of primordia is transported out of the central zone as opposed to moving with stem cells as they divide and differentiate (Galvan-Ampudia et al., 2020). Curiously, the pin1 inflorescence continues elongating and appears to maintain a stem cell pool, suggesting that auxin transport via PIN1 is not necessary for stem cell maintenance.

During initiation of primordia, ARF5 facilitates auxin-induced epigenetic reprogramming that activates a new long-term transcriptional program necessary for flower development. ARF5 recruits the chromatin remodelers BRAHMA (BRM) and SPLAYED (SYD), which are SWI/SNF ATPases, to target gene promoters to facilitate their transcriptional activation. The interaction between ARF5 and BRM/SYD can be blocked by IAA12, ensuring that remodeling only occurs when auxin is present in the primordia, preventing ectopic primordium initiation in the absence of auxin (Wu et al., 2015). Additionally, ARF5, as well as ARF3/4, directly and epigenetically facilitates STM repression during initiation of primordia (Chung et al., 2019). arf3/4 enhance the pin phenotype of the weak arf5 allele mp-S319, and the arf3/4/5 triple mutant shows an STM expression domain that is expanded into the peripheral zone. Furthermore, knocking down STM using miRNA in the triple arf mutant background restored primordium outgrowth, showing that this down-regulation is essential for floral primordium initiation and subsequent development. Curiously, ARF3 binds to regions of the STM promoter, while ARF5 down-regulation of STM was indirect. Lastly, ARF3/4 interact with the histone deacetylase HDA19, which associated with the STM locus to reduce levels the transcription-promoting epigenetic marker H3K27 there. This leads to epigenetic repression of STM in the primordia (Chung et al., 2019).

As previously mentioned, lateral organs are initiated at 137.5° angles from one another, following the Golden Ratio. Auxin’s role in establishing phyllotaxy has been well-established (Reinhardt et al., 2000, 2003). Two advances in auxin regulation of phyllotaxy are worth noting. First, arf3 mutants have defects in the phyllotaxy of flowers. ARF3 regulates the expression of transcription factors that establish the boundary between the meristem and lateral organs, specifically by repressing BOP1/2 and TEC3 and activating CUC1-3 (Zhang et al., 2022). The mis-regulation of these transcription factors in the arf3 background results in phyllotaxy defects. Additionally, other reports showed that ARF5 activates the expression of HISTIDINE PHOSPHOTRANSFER PROTEIN6 (AHP6), which encodes a cytokinin signaling inhibitor that regulates phyllotaxy by preventing concomitant organ initiation (Besnard et al., 2014a, b). Curiously, a recent study showed that DRNL cooperates with ARF5 to activate expression of AHP6, as well as CYTOKININ OXIDASE6 (CKX6), to reduce cytokinin accumulation to permit initiation of primordia (Dai et al., 2023).

Cell proliferation and outgrowth

Following floral primordium initiation, cells in the primordium must proliferate to allow it to grow away from the SAM. Little is known about how this phase of flower development is regulated. To date, the most direct experiment addressing the question of auxin and cell proliferation in the primordia showed that applying the auxin-transport inhibitor NPA to SAMs causes an increase in the expression of CYCLIND6;1 (CYCD6;1), though the role of CYCD6;1 in primordium formation is unclear (Bahafid et al., 2022, Preprint). There is another potential direct link from auxin to the cell cycle via the proposed auxin receptor and cell cycle activator SKP2A (del Pozo and Manzano, 2014; Powers and Strader, 2016). SKP2A is an F box protein, like the TIR1 auxin receptor, and auxin has been shown to promote its degradation. Additionally, the authors of the original SKP2A report propose that it regulates proteolysis of cell cycle factors in an auxin-dependent manner, suggesting a possible link from auxin perception to cell cycle activation, (Jurado et al., 2010).

As noted, CLAVATA signaling promotes auxin function during incipient primordium outgrowth. CRN, a pseudokinase, and CLV2, a LRR receptor-like kinase with only the extracellular domain, form a functional pair and repress stem cell proliferation independently of CLV1 (Willoughby and Nimchuk, 2021). CRN and CLV2 are expressed in incipient primordia, and mutants crn/clv2 show a terminated floral primordium phenotype in which floral primordia initiate properly but fail to grow out correctly, manifesting as bumps on the stem. These primordia terminate between stages 2 and 3 of flower development before the first floral organ primordia form, and primordium termination is independent of mis-regulation of floral meristem identity (Smyth et al., 1990; Jones et al., 2021). Floral primordium termination in crn is linked to a reduction in auxin signaling (DII) and transcriptional outputs (DR5). Expression of auxin biosynthesis genes, including YUCs, are decreased in crn SAMs, suggesting that the defects in crn mutants may be due to a reduction in auxin biosynthesis. Growing crn mutants in heat, in which endogenous auxin levels are increased, suppresses the termination phenotype as does mutating the heat sensor EARLY FLOWERING (ELF3), which results in a constitutive heat response in ambient temperatures (Delker et al., 2022). These data support a role for CRN as an activator of auxin function, though the precise mechanism for this remains to be elucidated (Jones et al., 2021). CLV1 can also promote outgrowth of primordia, but this function is normally masked by the inhibitory phosphatase POLTERGEIST (POL) (DeFalco et al., 2022; John et al., 2023). CLV1 acts non-cell autonomously from the OC, which is likely distinct from where CRN/CLV2 regulate outgrowth of primordia. Notably, this implies that stem cell signaling in the organizing center influences auxin levels in the primordia (John et al., 2023).

Other mutants showing a similar termination phenotype include lfy-6 ant-4 ail6-2 and rev phv phb ± (Prigge et al., 2005; Yamaguchi et al., 2013). The roles of LFY, AINTEGUMENTA (ANT), and AINTEGUMENTA-LIKE6 (AIL6) in promoting initiation and establishing floral meristem identity and their interactions with auxin have been well described; the latter will be discussed in the next section. The lfy-6 ant-4 ail6-2 triple mutant phenotype suggests that these transcription factors also promote outgrowth of primordia (Yamaguchi et al., 2013). Furthermore, genetic evidence suggests that LFY/ANT/AIL6, CLV2/CRN, and CLV3 promote auxin-dependent initiation of primordia together with auxin transport. Specifically, lfy pid double mutants, lfy ant ail-6 mutants treated with NPA, and heat-stressed clv2 yuc1/4 and clv3 yuc1/4 triple mutants all form pin inflorescences (Li et al., 2013; Yamaguchi et al., 2013, 2014b; Jones et al., 2021). It remains unclear whether these components regulate the early phases of flower development independently or concurrently. Finally, REV, PHV, and PHB, which belong to the class III Hd-ZIP transcription factor family and are expressed in incipient primordia, promote outgrowth of primordia. In the inflorescence meristem, class III Hd-ZIPs repress auxin-induced gene expression, while auxin activates expression of class III Hd-ZIPs, suggesting a potential link between the two in facilitating outgrowth of primordia (Prigge et al., 2005; Caggiano et al., 2017). Indeed, the HD-ZIPs have been proposed to promote outgrowth through the regulation of adaxial–abaxial patterning (Heisler and Byrne, 2020; Peng et al., 2022).

Floral fate establishment and reinforcement

As a floral primordium grows out, auxin contributes to the establishment of floral fate in the new lateral organ through ARF5-mediated activation of the transcription factor LEAFY (LFY). As there are several pathways through which ARF5 activates LFY, both directly and indirectly, this allows floral fate to be established and cemented throughout the development of the flower, preventing reversion to inflorescence identity. First, auxin activates ARF5-dependent transcription, which in turn activates LFY expression. ARF5 also activates expression of the transcription factors ANT/AIL6, which further promote LFY expression (Yamaguchi et al., 2013, 2016). This paper hypothesizes that an additional factor or factors activate LFY in addition to ARF5 and ANT/AIL6, and a recent preprint may have identified one of those factors as SHORTROOT (SHR), which promotes LFY expression after induction by ARF5, though direct binding of SHR to the LFY promoter has not yet been demonstrated (Bahafid et al., 2022, Preprint). It is also worth noting that, apparently independent of auxin function, SOC1 and AGL24 cooperate to activate LFY (Lee et al., 2008; Liu et al., 2008). Following this activation through these auxin-regulated transcription factor networks, LFY then activates APELATA1 (AP1), and together they promote floral meristem identity in the incipient primordia (Liljegren et al., 1999; Wagner et al., 1999).

LFY expression also requires BOP1/2 and PUCHI, a homolog of DRN/DRNL (Xu et al., 2010). Quintuple drn drnl puchi bop1/2 mutants mimic lfy phenotypes, and LFY protein levels in the SAM are reduced almost entirely (Chandler and Werr, 2017). An apparent contradiction is evident when considering the work discussed earlier that showed ARF5 represses DRN in the central zone, as one would expect that ARF5 should activate DRN/DRNL to facilitate the reinforcement of LFY expression in the primordia. Indeed, a recent report indicates that this may be the case: ARF5 activates DRNL in the peripheral zone, and together they activate ARF5 targets involved in initiation, outgrowth, and identity, such as AIL6, ANT, FIL, and TMO3. Furthermore, in the drnl mutant, ARF5 will activate expression of DRN in the peripheral zone to compensate for the loss of DRNL. Curiously, DRNL induction did not alter LFY expression, indicating that DRNL regulation of LFY may be indirect (Dai et al., 2023).

Another report suggests STM contributes to floral identity establishment first independently of AP1, a floral identity promoting transcription factor, and later by transcriptional activation of AP1 and UNUSUAL FLORAL ORGANS (UFO), which encodes an F-box protein that contributes to floral identity. STM expression resumes in a primordium once a groove separating the primordium from the SAM starts to form, at stage 2 (Smyth et al., 1990), indicating that it is expressed when the transcriptional program promoting floral fate is active. Genetic evidence suggests STM and AP1 act in parallel, as a weak stm allele enhances the floral fate defects of an ap1 mutant. Additionally, Smyth et al. (1990) show that in the AP1 domain, STM contributes to continued AP1 activation, providing a basis for STM reinforcement of floral fate. Finally, the authors also show that SHORT VEGETATIVE PHASE (SVP) and AGAMOUS-LIKE24 (AGL24), which encode transcription factors that promote inflorescence identity in primordia, are overexpressed in the AP1 domain when STM is repressed. This may suggest that STM repression of SVP and AGL24 may be one mechanism by which it influences floral fate (Roth et al., 2018). Additionally, STM promotes floral fate establishment by activating BOP2, which then activates LFY (Roth et al., 2018).

LFY also regulates the balance of hormones in incipient primordia to reinforce floral identity. While increased auxin levels promote the transition from axillary meristem identity to floral fate in incipient organs, gibberellic acid (GA) delays the transition of the meristem from producing axillary meristems to determinate floral meristems. LFY reduces GA levels in incipient floral primordia by activating the expression of the GA catabolic enzyme ELA1. This reduces GA levels in the primordia and allows floral identity to be established (Yamaguchi et al., 2014a).

Curiously, LFY seems to negatively regulate auxin accumulation while promoting auxin responses and transport. LFY represses expression of the auxin biosynthesis genes YUC4 and TAR2 in the primordia. Correspondingly, auxin levels are increased in a lfy mutant, indicating that LFY blocks auxin accumulation (Li et al., 2013). In contrast, the observations that the transcriptional response to auxin (DR5) is reduced in lfy mutants and that LFY overexpression results in increased DR5 in developing primordia indicate that LFY promotes auxin responses (Li et al., 2013). Additionally, LFY promotes auxin transport by promoting the functions of PIN1 and PINOID (PID), a kinase that promotes auxin transport by phosphorylating PINs (Li et al., 2013; Sauer and Kleine-Vehn, 2019). Another report suggests that LFY regulates PID function by activating its expression (Yamaguchi et al., 2013). These apparent contradictions in LFY regulation of auxin function and its effects on floral identity networks should be further explored but may suggest that LFY plays distinct temporal roles in auxin regulation during primordium formation.

Floral fate reinforcement

Unfortunately, little is known about how auxin may reinforce floral fate through the repression of inflorescence identity in the developing floral meristem. However, a few connections may be worth investigating in further detail. Several transcription factors that promote inflorescence identity, including SVP and AGL24, are expressed in incipient floral primordia. These repress expression of class B/C floral homeotic genes early in flower development, possibly to allow proliferation and outgrowth of the floral meristem before the development of specific floral organs (Liu et al., 2007; Gregis et al., 2009; Xu et al., 2010). SVP/AGL24 also reinforce floral meristem identity by promoting LFY expression, so it is likely that their function to promote inflorescence identity is transient (Grandi et al., 2012). As plants overexpressing SVP or AGL24 form flowers with mosaic inflorescence identity, it is crucial that these factors are down-regulated (Liu et al., 2007). The only link to auxin described thus far is the repression of ARF3, which promotes floral meristem identity, by SVP (Gregis et al., 2013). How SVP/AGL24 may regulate auxin function and how auxin function feeds back onto SVP/AGL24 activity to facilitate repression of inflorescence identity remain unknown.

Conclusion

Auxin influences both stem cell maintenance and initiation of floral primordia and thus likely facilitates crosstalk between incipient primordia and stem cells. Recent advances in our knowledge of stem cell regulation have shown that auxin both positively and negatively regulates stem cell proliferation; low levels of auxin response are required to maintain the stem cell pool in the long term, but temporary auxin depletion causes stem cell over-proliferation. Additionally, new work suggests that the primary source of auxin in the SAM appears to be floral primordia P2 and older, suggesting that auxin flux from primordia to the central zone may serve as a feedback mechanism regulating the balance between new floral primordium production and stem cell maintenance. Auxin also plays a crucial role in the subsequent stages of flower development by initiating new flowers, promoting outgrowth of primordia, and activating the subsequent establishment of floral identity. While auxin-dependent pathways regulating establishment of floral fate have been well-described, much less is known about the role of auxin in facilitating crosstalk between stem cell signaling and primordium initiation and outgrowth. Thus, future work could address the mechanisms by which SAM maintenance and organ production are intertwined. Several specific questions about these crosstalk mechanisms arise from advances in this topic. First, what are the mechanisms of crosstalk between auxin and transcription factors that influence stem cell maintenance? Second, what is the flow of auxin transport in the SAM required to initiate floral primordia, and how does auxin flux influence stem cell maintenance? Third, how does CLAVATA signaling promote auxin function to positively regulate stem cell maintenance? And lastly, what pathways promote outgrowth of floral primordia, and how does auxin interact with them? Addressing these questions will further clarify our understanding of how auxin dynamics in the SAM influence continual flower production essential for robust reproduction, allowing development of agricultural technologies that will ensure robust crop yields.

Conflict of interest

No conflict of interest declared.

Funding

The ZLN lab is supported by a National Institute of General Medical Sciences–Maximizing Investigators’ Research Award from the National Institutes of Health [R35GM119614]. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program to ES [DGE-2040435]. Any opinions, findings, and conclusions or recommendations expressed in this article are those of the authors and do not necessarily reflect the views of the National Science Foundation.