Update on Floral Organogenesis

Floral Organogenesis: When Knowing Your ABCs Is Not Enough1[OPEN] Bennett Thomson, Beibei Zheng, and Frank Wellmer* Smurfit Institute of Genetics, Trinity College, Dublin 2, Ireland ORCID ID: 0000-0002-2095-0981 (F.W.).

Flower development is one of the particularly wellestablished model systems for investigating the molecular and genetic mechanisms underlying organogenesis in plants. Over the past 30 years, this work has led to detailed insights into many of the cellular and developmental processes that occur during the formation of flowers. This progress was made possible especially by the identification and characterization of the floral organ identity factors, which specify the different floral organ types in a combinatorial manner. However, in recent years, the genes that act downstream of these master regulators have taken center stage because it has become increasingly clear that they execute many of the functions originally attributed to the floral organ identity factors. In this Update, we will summarize and discuss our current view of floral organogenesis with particular emphasis on recent progress in the field (see “Advances” box). We will briefly describe the latest models for floral organ identity factor function and outline open questions that need to be addressed to better understand how they act at the mechanistic level. Furthermore, we will discuss studies that have begun to reveal how a complex interplay of transcription factors, hormones, regulatory RNAs, and epigenetic modifiers controls different developmental processes during the formation of flowers. Last, we will outline recent efforts to better understand the evolution of flowers and venture a look ahead at likely future developments in the field of flowering research.

gene expression profiling experiments, in which the transcriptomes of developing (wild-type and mutant) flowers, individual types of floral organs, and different cell types were analyzed. These studies yielded a comprehensive view of gene expression during flower formation. However, because the available data did not always provide detailed information on when and where genes are expressed, additional studies conducted in recent years have aimed at increasing the spatio-temporal resolution of the gene expression map for flower development (Jiao and Meyerowitz, 2010; Wuest et al., 2010; Mantegazza et al., 2014; Chen et al., 2015; Ryan et al., 2015; Villarino et al., 2016). To this end, a wide range of methods, such as laser capture microdissection (Wuest et al., 2010; Mantegazza et al., 2014) or fluorescence-activated cell sorting, in combination with a flower synchronization system (Ó’Maoiléidigh et al., 2015; Villarino et al., 2016), was used. While most of this work has focused on a few model species, in particular, Arabidopsis

GENES INVOLVED IN FLORAL ORGANOGENESIS

Since the 1980s, forward and reverse genetic screens have led to the identification of dozens of key regulators of flower development (Causier and Davies, 2014; Ó’Maoiléidigh et al., 2014a, 2014b; Prunet and Jack, 2014). The functions of many of the genes have subsequently been studied in great detail using a wide range of experimental approaches. With the advent of the genomics era, this work has been complemented by

Work on flower development in our laboratory is funded by a grant from Science Foundation Ireland. B.Z. is supported by a postdoctoral fellowship from the Irish Research Council. * Address correspondence to [email protected]. [OPEN] Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.16.01288 1

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Update on Floral Organogenesis

(Arabidopsis thaliana) and rice (Oryza sativa), the development of next-generation DNA sequencing techniques has allowed the characterization of the floral transcriptomes of several other species of different families, such as woodland strawberry (Fragaria vesca; Hollender et al., 2014) and eucalyptus (Eucalyptus grandis; Vining et al., 2015) to name but a few. This work yielded valuable datasets not only needed to establish additional model plants for flowering research but also to better understand the genetic basis underlying the considerable differences in floral architecture found among the angiosperms (see below for further discussion). The results of the studies described above showed that flower development involves the differential expression of thousands of genes, probably corresponding to as much as one-quarter of all the genes in a plant’s genome (Ryan et al., 2015). Because many of these genes are known or predicted to have regulatory functions, it appears that the gene networks that control floral organogenesis are exceptionally complex. However, attempts to systematically characterize the functions of these differentially expressed genes have not been met with much success in spite of the powerful reverse genetics approaches available, especially for Arabidopsis. One reason for this may be that many of these genes play minor roles in flower formation so that their inactivation does not lead to readily discernable mutant phenotypes. Furthermore, the high degree of functional redundancy found among (often closely related) genes

in plants likely hampers their analysis. With the invention of sophisticated genome editing approaches such as CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated; Wright et al., 2016), the latter genes can now be studied more efficiently by generating full loss-of-function alleles and by subsequently creating lines with multiple gene knockouts. Thus, many additional regulators of flower development will likely be discovered in the near future, and this will lead to an increasingly complete view of the gene sets that control floral organogenesis. FLORAL ORGAN SPECIFICATION

The ABC model of floral organ identity specification (Coen and Meyerowitz, 1991) has guided research on flower development over the past 25 years. The experimental basis, the history, as well as the modifications and extensions of the original model have been described in several recent reviews (Bowman et al., 2012; Ó’Maoiléidigh et al., 2014a, 2014b; Prunet and Jack, 2014; Wellmer et al., 2014b; Sablowski, 2015). In brief, the model proposes that the activities of certain combinations of floral organ identity genes, which encode transcription factors of the MADS domain family, lead to the formation of sepals, petals, stamens, and carpels (Fig. 1, A and B). Although specific aspects of the model (in particular, the concept of “A function”) have been put into doubt in recent years (Litt, 2007; Causier et al., 2010; Wollmann et al., 2010), its overall

Figure 1. Molecular aspects of the ABCE model. A, Illustrations of the organs found in the outer to inner (left to right) whorls of the flower. Se, Sepal; Pt, petal; St, stamen; Ca, carpels. B, The ABCE model of flower development (Theissen, 2001). Specific classes of floral organ identity genes are active within each floral whorl. A class genes specify sepals in the first whorl; A and B class genes specify petals within the second whorl; B and C class genes specify stamens within the third whorl; C class gene function specifies carpel identity within the fourth whorl. The E class genes are active within all four whorls. C, Combinatorial interactions of floral organ identity factors within each whorl form dimeric (not shown) and higher-order tetrameric complexes. D, It has been proposed (Pajoro et al., 2014) that the floral organ identity factors can act as pioneer factors, influencing chromatin accessibility throughout flower development. E, Higher-order MADS domain complexes likely interact with transcriptional cofactors and chromatin remodelers to regulate target gene expression. The complexes are predicted to induce short-range DNA looping by binding adjacent CArG box sequences (black rectangles) present within cis-regulatory elements (see text for details). Plant Physiol. Vol. 173, 2017

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validity across diverse angiosperm species is unquestioned. The molecular basis of the ABC model is summarized in the “quartet model” (Theissen, 2001; Fig. 1C), which posits the existence of four regulatory complexes, each composed of four floral organ identity factors, that bind to two cis-regulatory elements [containing so-called CArG boxes; consensus: 59-CC(A/T)6GG-39] in the promoter of a gene to induce DNA looping in-between the two binding sites. Each of these complexes was predicted to contain “E function” cofactors, which also belong to the family of MADS domain proteins. Both the in vivo existence of the floral organ identity factor complexes (Smaczniak et al., 2012) as well as the occurrence of DNA looping (Melzer et al., 2009; Mendes et al., 2013; Jetha et al., 2014) have been experimentally confirmed in recent years. Despite this considerable progress in our understanding of the floral organ identity factors, key questions about their functions remain unsolved. In particular, it is currently not known how the MADS protein complexes act at the mechanistic level. The different floral organ identity factors have similar albeit not identical DNA binding specificities (Muiño et al., 2014), and the results of genomewide localization studies (Kaufmann et al., 2009, 2010; Wuest et al., 2012; Ó’Maoiléidigh et al., 2013; Pajoro et al., 2014) showed that their global binding patterns exhibit large overlaps even in cases where two factors are not part of the same complex. At the same time, the sets of bona fide target genes, i.e. genes that are bound and respond to a perturbation of the different floral organ identity factors, are largely distinct (Yan et al., 2016). Furthermore, the expression of the majority of genes bound by the floral organ identity factors is not, or only slightly, altered upon binding by the MADS protein complexes (Kaufmann et al., 2010; Wuest et al., 2012; Ó’Maoiléidigh et al., 2013). Thus, it appears possible that a large number of their binding sites in the genome are nonfunctional at least in the context of floral organ identity specification (i.e. they might be functional in other processes during plant development when they are bound by additional members of the large MADS domain protein family). Taken together, these observations led to the question of how the specificity of the different floral organ identity factor complexes is controlled. Current models of transcription factor function in eukaryotes (Slattery et al., 2014) suggest that this control of specificity might be based on a number of contributing factors. These could include the spatial arrangement of binding sites within larger cis-regulatory modules and differences in the affinities of the transcription factors to specific sites (“strong” binding sites are thought to more likely trigger differential expression than “weaker” sites; Slattery et al., 2014). Also, the presence or absence of additional transcriptional regulators, which cooperatively or as part of a transcription factor collective (Junion et al., 2012) act with the MADS domain proteins on a target gene promoter, may determine whether a gene is activated or repressed. The results of a recent proteomics study strongly suggest that the floral organ identity factor complexes indeed associate with other transcription factors as well as 58

with proteins involved in the epigenetic control of gene expression (Smaczniak et al., 2012; Fig. 1E). This idea is further supported by studies that specifically analyzed the interaction of the MADS domain proteins with other transcription factors, such as members of the Auxin Response Factor family (Ripoll et al., 2015) to name only one recent example. Because it was found that DNA binding by the floral organ identity factors often precedes an increase in DNA accessibility at target gene promoters, it has been suggested that they may act as pioneer factors (Pajoro et al., 2014; Fig. 1D). Such pioneer factors bind to condensed chromatin and then alter chromatin conformation through the recruitment of epigenetic modifiers to facilitate or prevent binding by other transcriptional regulators (Zaret and Mango, 2016). Whatever the precise mechanism of floral organ identity factor function, it is clear that these conserved master regulators have played a central role in the evolution of floral structures (e.g. Soltis and Soltis, 2014; Gramzow and Theissen, 2015; Becker, 2016; Silva et al., 2016). In fact, it appears likely that differences in the sets of target genes for a given factor can explain many of the alterations in floral morphologies found among the angiosperms. At the same time, it would be important to identify, for each of the different classes of floral organ identity factors, core sets of targets that are shared between related species or even between angiosperms belonging to distinct families. These genes could provide the information necessary for establishing the basic blueprint for the different types of floral organs. While such studies have not yet been conducted systematically, the first steps in this direction have already been taken (Muiño et al., 2016) and will likely lead to much activity in this area in years to come.

GENES ACTING DOWNSTREAM OF THE FLORAL ORGAN IDENTITY FACTORS

How do the floral organ identity factor complexes control organ morphogenesis? García-Bellido proposed in the mid-1970s that master regulators (“homeotic selector genes”) such as the floral organ identity factors directly control the expression of (“realizator”) genes that are required for the terminal differentiation of organs (GarcíaBellido, 1975). That this idea is at least in part correct for the floral organ identity factors is suggested by the expression of the corresponding genes in floral organs throughout most of their morphogenesis (Ó’Maoiléidigh et al., 2014a, 2014b). Also, the results of perturbation experiments, in which the floral organ identity genes were specifically disrupted or activated at different floral stages, showed that these master regulators have crucial functions in processes that take place during intermediate and late stages of flower development (Ito et al., 2007; Wuest et al., 2012; Ó’Maoiléidigh et al., 2013). However, the now prevailing view is that they largely act by controlling the expression of other genes with regulatory functions. This conjecture is most strongly supported by the results of Plant Physiol. Vol. 173, 2017

Update on Floral Organogenesis

studies (which were already briefly mentioned above) in which the target genes and gene expression programs acting downstream of the floral organ identity factors were identified on a genome-wide scale (Kaufmann et al., 2009, 2010; Wuest et al., 2012; Ó’Maoiléidigh et al., 2013; Pajoro et al., 2014). These studies not only showed that each factor has at least a few hundred direct target genes and that these sets of targets exhibit considerable changes during the course of flower development, but also that they are highly enriched for genes with regulatory functions. The expression of these targets can be both activated and repressed, indicating that the floral organ identity factor complexes are bifunctional in their activity. The systematic identification of the genes acting downstream of the floral organ identity factors triggered the question how these genes control the plethora of processes that take place during floral organ morphogenesis. Work conducted in recent years has led to several examples where we now understand in some detail the contributions to floral organogenesis of individual gene regulatory events that occur in response to floral organ identity factor activity. We will discuss a few of these cases in the following section.

CONTROL OF FLORAL ORGAN GROWTH AND DEVELOPMENT

An early example for a gene that is directly controlled by floral organ identity factors and that regulates an essential process during floral organ development is NOZZLE/SPOROCYTELESS (NZZ/SPL), a master regulator for the formation of sporogenic tissues in Arabidopsis (Schiefthaler et al., 1999; Yang et al., 1999). It was shown that once activated by the C function regulator AGAMOUS (AG), which is involved in the specification of the reproductive floral organs, the NZZ/SPL transcription factor can trigger microsporogenesis in the absence of AG activity (Ito et al., 2004). Another example for a transcription factor-coding gene that acts downstream of floral organ identity factors in Arabidopsis is JAGGED (JAG). JAG, together with its paralog NUBBIN (NUB), controls the growth of leaf margins and floral organs, especially in their distal regions (Dinneny et al., 2004, 2006; Ohno et al., 2004). Both genes have been shown to be regulated by B, C, and E class floral organ identity factors (Gómez-Mena et al., 2005; Kaufmann et al., 2009; Wuest et al., 2012; Ó’Maoiléidigh et al., 2013). Using computer modeling, and petal growth as an example, JAG was proposed as a possible distal-specific growth factor (Sauret-Güeto et al., 2013). Furthermore, through three-dimensional analysis of cell geometry and DNA synthesis, it was shown that JAG promotes changes in growth patterns when organ primordia are initiated from a (floral) meristem and that it coordinates cell growth with the cell cycle (Schiessl et al., 2012). Thus, it appears that JAG/NUB play roles in both organ patterning and growth control, possibly mediating between these two processes. The idea of a more complex role for JAG in Plant Physiol. Vol. 173, 2017

lateral organ development is supported by the results of experiments that aimed at the genome-wide characterization of JAG target genes (Schiessl et al., 2014). Among the targets identified were regulators of diverse processes, including meristem development and organ growth, tissue polarity, cell wall modification, and cell cycle progression. Additional experiments showed that JAG directly represses the KIP RELATED PROTEIN4 (KRP4) and KRP2 genes, which control the transition to the Synthesis (S)-phase of the cell cycle, and that krp2 and krp4 mutant alleles largely suppressed the jag mutant phenotype (Schiessl et al., 2014). Thus, JAG appears to regulate organ growth, at least in part, through direct cell cycle control. A third example that illustrates the complexity of the gene regulatory networks downstream of the floral organ identity factors involves the Arabidopsis gene RABBIT EARS (RBE), which encodes a zinc-finger domain-containing transcription factor that mediates the control of petal development (Takeda et al., 2004). RBE has been shown to act directly downstream of the B function regulators APETALA3 (AP3) and PISTILLATA (PI; Wuest et al., 2012), which are involved in the specification of petals and stamens. During petal initiation, RBE controls the microRNA miR164-dependent pathway (Huang et al., 2012), which mediates boundary formation in-between organ primordia via regulation of CUP-SHAPED COTYLEDON genes (Sieber et al., 2007). During this initial phase of petal development, RBE was found to repress the expression of certain TCP transcription factor-coding genes (Huang and Irish, 2015), which are thought to control the transition from cell division to cell expansion and differentiation during organ growth (Huang and Irish, 2016). This repression is lost or reduced as petal development progresses (Huang and Irish, 2015), leading to the promotion of growth and differentiation, thus contributing to the formation of mature petals. The floral organ identity factors also control the expression of many genes involved in the metabolism of or the response to different phytohormones, including auxin, cytokinins, gibberellins, and jasmonic acid. In fact, these hormones have been shown to mediate diverse processes during flower development, ranging from the control of meristematic activity, to pattern formation and organ maturation (Chandler, 2011). How exactly these hormones act during floral organogenesis is in many cases still unknown, however significant progress toward a better understanding of their functions has been made in recent years, for example, by studying and comparing the patterns of auxin and cytokinin signaling at different stages of gynoecium development (Marsch-Martínez et al., 2012). Such work has begun to address the extensive cross talk (and in some cases, antagonism) known to exist between the different hormone signal transduction pathways, and future research will undoubtedly aim at elucidating this interplay at the molecular level. Taken together, the examples discussed above illustrate the impressive progress that has been made in recent years in understanding the molecular functions of key regulators of floral organogenesis, which act 59

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directly or indirectly downstream of the floral organ identity factors. Given that the number of regulatory genes with known or presumed roles in flower development is very large (see above), it will in all probability take many more years before we will have obtained a comprehensive view of the regulatory events that take place during floral organogenesis. Thus, the development of new experimental methods and protocols that allow a more high-throughput characterization of floral regulators is urgently needed to expedite the systematic analysis of the gene network underlying floral organogenesis.

CONTROL OF FLORAL MERISTEM DETERMINACY

In contrast to shoot apical meristems, which are indeterminate, floral meristems cease their activity once the formation of all floral organs has been initiated (at around stage 6 in Arabidopsis; stages according to Smyth et al., 1990). Molecularly, this loss of meristematic activity is often monitored by the expression of WUSCHEL (WUS), which encodes a homeodomain-containing transcription factor that functions as an important regulator of stem cell fate (Mayer et al., 1998). How is the determinacy of floral meristems regulated? It was shown 15 years ago that the floral organ identity factor AG, which is expressed in the center of the floral meristem from around stage 3, is essential for terminating the expression of WUS in Arabidopsis (Lenhard et al., 2001; Lohmann et al., 2001). This result explained the phenotype of ag mutant flowers, which not only show organ identity defects (i.e. an absence of reproductive floral organs) but also a strong overproliferation of the floral meristem (Yanofsky et al., 1990). Also, it was found that other floral regulators involved in the control of floral meristem determinacy, such as PERIANTHIA (Das et al., 2009; Maier et al., 2009), act upstream of AG. Thus, the floral meristem defects of these mutants are in all probability a consequence of AG misregulation. So how does AG repress WUS? It has been proposed that this occurs through both direct and indirect mechanisms (Fig. 2). It was shown that AG binds to CArG box sequences in the WUS promoter and recruits a Polycomb group (PcG) complex involved in the stable silencing of gene expression through covalent histone modifications (Liu et al., 2011). As a consequence of this direct regulation, WUS expression declines over time. Indirect mechanisms are then required to terminate WUS expression immediately after all floral organ primordia have been initiated. The KNUCKLES (KNU) transcription factor (Payne et al., 2004) has been shown to play a key role in this process. KNU is directly activated by AG at stage 6, exactly at the time when floral stem cell activity ceases (Sun et al., 2009). KNU then represses WUS likely through direct regulation. The timing of the induction of KNU by AG has been shown to be a consequence of PcG protein eviction from the KNU promoter by AG, which over time (and coupled to cell division cycles) leads to a removal of repressive chromatin marks and an activation of KNU expression (Sun et al., 2014). 60

Figure 2. Regulation of floral meristem determinacy. In Arabidopsis, stem cell activity within the developing flower ceases around stage 6. WUS is down-regulated by several pathways within the floral meristem, resulting in floral meristem termination (see the main text for details). Solid lines and dashed lines indicate direct and indirect regulation, respectively. “X” denotes a proposed unidentified factor whose activity is regulated by FHY3.

Despite the recent progress in our understanding of the molecular mechanisms underlying the control of floral meristem determinacy, several open questions remain. For example, why is the indeterminacy phenotype of knu mutant flowers much weaker than that observed for ag? One possibility is that other regulators act in parallel with KNU. A good candidate for this is the regulator of carpel and nectary development CRABS CLAW (CRC). Crc mutant flowers show mild determinacy defects, which are much enhanced when AG activity is reduced (Bowman and Smyth, 1999). Also, like KNU, CRC is directly activated by AG (Gómez-Mena et al., 2005; Ó’Maoiléidigh et al., 2013), and its expression commences at stage 6 as well (Bowman and Smyth, 1999). Thus, it is tempting to speculate that the activation of CRC by AG may involve the same PcG protein eviction mechanism identified for KNU. However, one key difference between KNU and CRC is their expression patterns. While early KNU expression is found in the center of the floral meristem, and especially in the organizing center where WUS is expressed (Sun et al., 2009), CRC expression occurs on the abaxial side of emerging carpel primordia (Bowman and Smyth, 1999). Thus, the activation of these genes by AG in neighboring but nonoverlapping domains of the flower must involve additional spatial inputs. Plant Physiol. Vol. 173, 2017

Update on Floral Organogenesis

In addition to the AG-dependent pathway, other regulatory genes have been identified that control floral organ determinacy. They include SUPERMAN (SUP; Sakai et al., 1995), which encodes a transcription factor closely related to KNU, JAG, NUB, and RBE (Sakai et al., 1995). Sup mutants have supernumerary stamens and exhibit an increase or decrease of (abnormal) carpel tissue, depending on the allele. When combined with mutant alleles for AG, mutations in sup lead to a dramatic meristem overproliferation phenotype (Bowman et al., 1992), suggesting that AG and SUP function in parallel pathways. It has been suggested that SUP is involved in controlling the boundary between the third and fourth floral whorls, possibly by repressing the expression of the B function regulators AP3 and PI (Sakai et al., 1995). Furthermore, it has been shown that SUP controls cell proliferation and differentiation in a hormone-dependent manner (Nibau et al., 2011). However, how SUP acts to control floral meristem determinacy is currently unknown. Another, unexpected, regulator of floral determinacy was described in a recent study. The transcription factor FAR-RED ELONGATED HYPOCOTYL3 (FHY3), which is involved in photomorphogenesis, was shown to control the expression of the stem cell regulator CLAVATA3 (CLV3) in the shoot apical meristem (Li et al., 2016). In floral meristems, FHY3 controls determinacy by directly repressing CLV3 and by activating the E class gene SEPALLATA2. In summary, the results of the studies discussed above show that the genetic mechanisms that regulate floral meristem determinacy are surprisingly complex, and likely evolved to allow plants to precisely control the timing of stem cell termination and to tightly coordinate this process with the early development of floral organ primordia.

EVOLUTIONARY HISTORY OF FLORAL ORGANS

von Goethe famously proposed more than 200 years ago that floral organs are in essence modified leaves (von Goethe, 1790). While it is now well established that the activities of the floral organ identity factors are necessary and sufficient for this transformation process (Goto et al., 2001; Ditta et al., 2004), it is still unclear how exactly they suppress leaf development or alter it so that floral organs can form. The genome-wide studies mentioned above clearly showed that the floral organ identity factors activate many genes with flower-specific expression, suggesting that these large gene sets provide the specialized functions needed to guide much of floral organogenesis. What is not yet known is what happens to the genetic program for leaf development upon the initiation of floral organ primordia. The analysis of lines with mutations in the different classes of floral organ identity genes showed that floral organs were converted into leaf-like structures (Goto et al., 2001; Ditta et al., 2004), suggesting that the genetic program for leaf development is still active or alternatively can readily be reactivated when the flowerspecific inputs are abolished. However, how the floral Plant Physiol. Vol. 173, 2017

organ identity factors utilize and/or suppress the gene network controlling leaf development to mediate floral organ morphogenesis is currently not well understood. While sepals (and to some extent also petals) still morphologically very much resemble leaves, the reproductive floral organs are more complex and appear to be highly derived. However, upon closer examination, the resemblance between leaves and floral organs can clearly be seen even here. For example, the carpels of Arabidopsis gynoecia, which fuse to engulf the ovary, still exhibit leaf-like features, such as stomata, and in some species carpel valves even carry trichomes. Also, many regulatory genes that were first identified based on their contribution to leaf development are also active in flowers. For example, regulators of organ polarity such as KANADI1 (KAN1; Kerstetter et al., 2001), by and large, exhibit similar expression patterns in floral organs and leaves (in the case of KAN1, on the abaxial side of the developing organs). Therefore, a central question that needs to be addressed to better understand the origins of flowers is to what extent the formation of floral organs relies on the genetic program for leaf development and how this program was altered over evolutionary time to bring about floral organogenesis.

OUTLOOK

The results of the studies discussed above, as well as many other important contributions not mentioned here due to space constraints, have led in recent years not only to an ever more detailed view of floral organogenesis but also to real fundamental breakthroughs in our understanding of this key process in plant development. In fact, the amount of available data is now so large that even noted experts in the field will struggle to memorize, process, and integrate it all. This problem has been addressed, at least in part, through the development of data repositories such a FLOR-ID, a handcurated database containing information from more than 1,600 research papers on flowering (Bouché et al., 2016). By providing a wealth of information at the tip of a finger, such databases will undoubtedly aid in deciphering the composition and architecture of the gene networks that control floral organogenesis. In spite of the progress that has been made in the field of flower development in recent years, many open questions remain to be answered. Several of them have already been mentioned above, and many others have been summarized in recent reviews (Scutt and Vandenbussche, 2014; Wellmer et al., 2014a; see also “Outstanding Questions” box). The ability to address and ultimately solve such open questions in biology often depends on the development of novel technologies that lead to new experimental approaches, and researchers in the flowering field have always quickly adopted new methods whenever they appeared pertinent. For example, the use of advanced life imaging techniques has yielded unprecedented insights into the cellular and mechanical dynamics during floral organogenesis (Hervieux et al., 2016; Prunet et al., 2016). 61

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ACKNOWLEDGMENTS We thank our colleagues and collaborators for their input and contributions. We sincerely apologize to those colleagues whose work we could not cite or discuss here due to space constraints. Received August 16, 2016; accepted October 24, 2016; published October 27, 2016.

LITERATURE CITED

Also, computational models of key aspects of flowering, such as floral meristem patterning (Alvarez-Buylla et al., 2010) or the establishment of organ primordium polarity (La Rota et al., 2011), have led to a framework that can guide and drive further experimentation. Progress in technology development, in particular, advanced DNA sequencing methods, will also greatly facilitate already ongoing efforts (for summary, see Chang et al., 2016) to establish additional model plants for flowering research. The establishment of such additional models is of pivotal importance not only because they may provide information on the reproductive biology of a wide range of agriculturally important plants but also because they will allow us to better address the evolutionary mechanisms that act on flower development. A recent study (Wang et al., 2015), which focused on floral organ specification in Nigella damascena (Ranunculaceae), a plant with a spiral instead of a whorled arrangement of floral organs, demonstrated the relative ease with which such new models can now be established. However, it also showed how valuable (and indeed necessary) the data obtained from the “classic” model plants of flowering research are for such an endeavor. Therefore, a strong case can be made to continue a focus on flower development in Arabidopsis, rice, and other well-established models. However, research in the field will have to significantly broaden to include other angiosperm species of diverse families to obtain a truly comprehensive view of the genetic and cellular mechanisms controlling floral organogenesis. 62

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Update on Floral Organogenesis

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