Protoplasma DOI 10.1007/s00709-015-0812-7

REVIEW ARTICLE

Arabidopsis flower development—of protein complexes, targets, and transport Annette Becker 1 & Katrin Ehlers 1

Received: 20 March 2015 / Accepted: 23 March 2015 # Springer-Verlag Wien 2015

Abstract Tremendous progress has been achieved over the past 25 years or more of research on the molecular mechanisms of floral organ identity, patterning, and development. While collections of floral homeotic mutants of Antirrhinum majus laid the foundation already at the beginning of the previous century, it was the genetic analysis of these mutants in A. majus and Arabidopsis thaliana that led to the development of the ABC model of floral organ identity more than 20 years ago. This intuitive model kick-started research focused on the genetic mechanisms regulating flower development, using mainly A. thaliana as a model plant. In recent years, interactions among floral homeotic proteins have been elucidated, and their direct and indirect target genes are known to a large extent. Here, we provide an overview over the advances in understanding the molecular mechanism orchestrating A. thaliana flower development. We focus on floral homeotic protein complexes, their target genes, evidence for their transport in floral primordia, and how these new results advance our view on the processes downstream of floral organ identity, such as organ boundary formation or floral organ patterning.

Keywords Arabidopsis thaliana . Flower development . Floral homeotic genes . Protein complexes . Protein transport . MADS-box protein target genes

Handling Editor: David Robinson * Annette Becker [email protected] 1

Institute of Botany, Justus-Liebig University, Heinrich-Buff-Ring 38, 35392 Gießen, Germany

Flower meristem dynamics—a primer Flowers are among the most beautiful examples for mutually beneficial relationships between animals and plants. Even though most flowers are composed of only four different types of organs, their variability in structure, color, and scent is astonishing and fine-tuned for attracting insect or bird pollinators or to allow effective wind pollination. All floral organs arise from a floral meristem which, unlike the shoot or root meristem, terminates its stem cell activity once all floral organs are formed. We provide here a short introduction into flower meristem formation, activity, and termination and point to other recent reviews which provide more comprehensive insights into these mechanisms (Holt et al. 2014; Lifschitz et al. 2014; Chandler 2012). Genetic and biochemical studies have shown that a vegetatively growing Arabidopsis thaliana plant encountering conditions favorable for flowering, such as a matching photoperiod, expresses the FLOWERING LOCUS T (FT) protein in its rosette leaves. FT is then transported through the phloem toward the shoot apex and activates, together with FLOWERING LOCUS D (FD) the expression of APETALA1 (AP1) and LEAFY (LFY) which are required for floral meristem identity (Corbesier et al. 2007; Weigel et al. 1992; Wigge et al. 2005). Both proteins act in a complex to finally activate the floral homeotic genes which are required for the organ identity of the vegetative sepals and petals and the reproductive stamens and the gynoecium (the male and female organs, respectively). The floral meristem releases floral organ primordia at its flanks, and the number and position of stem cells are tightly regulated ensuring meristem stability as well as allowing cells to accumulate before organogenesis. Thus, the meristem is a defined structure even though its constituent cells are constantly changing. Cells exiting the meristem adopt their future cell fate according to their position within the meristem, such

A. Becker, K. Ehlers

that they remain in their original cell layers. The homeodomain-containing genes WUSCHEL (WUS) und SHOOT MERISTEMLESS (STM) function synergistically during shoot, inflorescence, and floral meristem development and maintenance. In wus and stm mutants, the floral meristem ceases prematurely because a smaller number of stem cells in the central zone are formed which cannot keep up with the production of a larger number of cells in the periphery required for floral organ formation (Lenhard et al. 2001; Clark et al. 1996; Laux et al. 1996; Long et al. 1996; Endrizzi et al. 1996). Conversely, overexpression of WUS leads to a conversion of cells in organ primordia into cells with stem cell characteristics (Gallois et al. 2004). WUS activation is limited by the CLAVATA (CLV) signaling loop where the small apoplastic signaling peptide CLV3, activated by WUS in L1 and L2, moves toward L3 where it binds to the CLV1 and CLV2 receptors and other receptor-like kinases to indirectly repress WUS expression (Kinoshita et al. 2010). When any of the CLV genes are mutated, the meristem’s central zone increases due to an overexpression of WUS (Fletcher et al. 1999; Brand et al. 2000). WUS also directly activates the floral homeotic gene AGAMOUS (AG) by direct binding to its second intron, and thus, WUS regulates not only stem cell maintenance but is also required for floral organ initiation (Lohmann et al. 2001). Once the floral meristem has produced all floral organ primordia, it is consumed in the process of carpel development and stem cell activity ceases. For this process, the floral homeotic C function gene AG is required to repress WUS by directly binding to its genomic locus and by the recruitment of Polycomb group proteins that methylate chromatin to silence the WUS genomic locus (Lenhard et al. 2001; Liu et al. 2011). In addition, AG activates KNUCKLES (KNU) by replacing repressing histone marks in a time-dependent manner, and KNU then represses WUS expression (Sun et al. 2014). Also, the floral homeotic A function gene APETALA2 (AP2), which also promotes stem cell fate in the floral meristem, is translationally repressed by miR172d to achieve floral determinacy (Chen 2004). Moreover, additional transcription factors (TFs), such as SUPERMAN (SUP), CRABS CLAW (CRC), PERIANTHA (PAN), and ULTRAPETALA (ULT), also regulate floral meristem maintenance and termination, but to a lesser extent and in a partly redundant way, suggesting a high level of genetic robustness in the floral meristem termination network (Carles et al. 2005; Maier et al. 2011; Prunet et al. 2008).

Setting floral organ identity The genes underlying floral organ identity specification in Arabidopsis thaliana (thale cress), Antirrhinum majus (snapdragon), Petunia hybrida (petunia), Oryza sativa (rice), and a limited number of other genetic model plants have been

identified and intensely studied during the past 20 years (see recent reviews by Wellmer et al. 2014; Yoshida and Nagato 2011; Causier and Davies 2014; Bowman et al. 2012). With the exception of AP2, all floral homeotic proteins are MADSbox TFs. In A. thaliana, the A function proteins alone specify sepal organ identity, A and B function together specify petal identity, B and C function proteins together specify stamen organ identity, and C function alone carpel identity. In addition, the E function is required for the formation of all floral organs, and finally, the D function specifies the ovules within the carpel. The A function proteins of A. thaliana are encoded by AP1 and AP2, B function proteins are encoded by APETALA3 (AP3) and PISTILLATA (PI), the C function is encoded by AG, and the combinatorial action of SEEDSTICK (STK), SHATTERPROOF1, and 2 (SHP1, SHP2) is required for the D function and the SEPALLATA1-4 (SEP1-4) genes carry out the E function in a largely redundant way. The MADS domain TFs act in higher order complexes to activate downstream target genes (Theißen and Saedler 2001; Bowman et al. 1991; Pelaz et al. 2000; Pinyopich et al. 2003; Ditta et al. 2004). Extensive genetic cross talk occurs between the floral homeotic proteins as they regulate target genes and their own expression, e.g., the C function is repressed in the first two floral whorls by A function proteins and vice versa. This is realized by balancing A and C function expression involving miR172 (Wollmann et al. 2010). The floral homeotic functions of AP1 and AP2, an AP2/EREPB TF, are not found outside the closer relatives of A. thaliana suggesting that they underwent rather recent neofunctionalization, but their role in floral meristem specification may be more ancestral (Litt and Irish 2003). Orthologs of the A. thaliana floral homeotic genes have now been analyzed in many species, including not only core eudicots, but also monocots like Oryza sativa (rice), Zea mays (corn), or basal eudicots like the Ranunculales species Eschscholzia californica (California poppy) (Lange et al. 2013; Mena et al. 1996; Yamaguchi et al. 2006; Yellina et al. 2010). Observation of phenotypes caused by loss of function mutants or by virusinduced gene silencing (VIGS) allows for a comparison of floral homeotic programs across a wide range of species with an increasing number of species that become amenable to functional approaches (Lange et al. 2013). Many of these studies show that the principal players of the genetic network orchestrating floral organ initiation have functions comparable to their orthologs in A. thaliana. However, as segmental or whole genome duplications have arisen several times independently throughout the angiosperms, copy numbers for the floral homeotic genes differ from species to species, and many paralogous pairs underwent subfunctionalization (Mena et al. 1996; Yamaguchi et al. 2006). In the following, we will concentrate on recent discoveries on the formation

Arabidopsis flower development review

of floral homeotic complexes, their targets, and evidence on their noncell autonomous action in A. thaliana.

Symplasmic transport of flower developmental regulators Several TFs involved in the regulation of flower development, such as LFY, AG, WUS, and STM, act in a noncell autonomous manner. They migrate symplastically through plasmodesmata (PDs) — the cytoplasmic cell connections in plants — into neighboring cells to coordinate developmental processes outside their mRNA expression domain (e.g., Han et al. 2014; Vaddepalli et al. 2015). Most likely, TF movement and noncell autonomous activity is spatiotemporally controlled by developmental changes of the PD number, opening state, and functional capacities (Burch-Smith et al. 2011; Ehlers and Große Westerloh 2013). Compared to other plant organs, however, PD networks in floral organs have only seldom been investigated, and our knowledge on the biological relevance of PD transport during flower development is scarce. Some noncell autonomous transcription factors (NCATFs) like LFY spread cell-to-cell in a nonselective manner by simple diffusion, as shown with GREEN FLUORESCENT PROTEIN (GFP)-tagged LFY constructs with a size of 74 kDa (Wu et al. 2003). This transport mode does not seem to require direct interaction with PD components and is limited by the PD aperture (denoted as size exclusion limit, SEL) which is developmentally controlled and reaches values of up to 81 kDa in meristematic tissues (Kim et al. 2005a). However, the biological relevance of LFY acting as a PD-mobile NCAT F to establish floral meristem identity is questionable, since LFY is expressed in all three cell layers of the floral meristem during the very early stages of flower development (Weigel et al. 1992; Blázquez et al. 1997). We hypothesize that the symplastic PD transport of LFY and other NCATFs within their domains of gene expression may provide the means for a strict synchronization of the cells within their developmental domain and possibly represents a fail-safe mechanism against stochastic fluctuations in gene expression or mutations rendering patchy expression. Targeted PD transport is accompanied by an enlargement of the SEL (Lucas et al. 1995) and requires protein-protein interactions between PDs and NCATFs, but the mechanism is poorly understood (Wu and Gallagher 2012). It is known that WUS and STM require their respective homeodomain for PD transport (Kim et al. 2005b; Daum et al. 2014), but NCAT Fs do not seem to share a common PD-targeting motif, since they are broadly distributed across distinct protein families and comprise WOX- and KNOX-homeodomain TFs, GRAS domain TFs, MADS box TFs, members of the CETS gene family, and Myb-like DNA-binding domain proteins (e.g., Rim et al. 2011; Chen et al. 2013). Cell- and tissue-specific

capacities for targeted PD transport may rely on the occurrence of specific transport facilitators like chaperones (Xu et al. 2011) or on the respective PD-protein equipment at the diverse cell interfaces, which may be decisive for the recognition of individual PD-targeting motifs. Different members of the PLASMODESMATA-LOCATED PROTEIN1 family have, e.g., been shown to occur in the distinct zones of inflorescence meristems and floral primordia (Bayer et al. 2008).

Transport of floral homeotic proteins during floral organ formation Information on the noncell autonomous action of A. thaliana floral homeotic MADS box proteins comes from the comprehensive studies of Urbanus et al. (2009, 2010), who compared previously described MADS gene expression patterns with the distribution and PD mobility of the MADS proteins (27– 28 kDa, Wu et al. 2003). They investigated GFP-tagged versions of AP3, PI, AG, and SEP3, which were all expressed from the MERISTEMLAYER1 promoter (pAtML1) in wildtype plants (Urbanus et al. 2010; flower stages according to Smyth et al. 1990). All constructs caused mild overexpression phenotypes in the flower, and they all were able to move symplastically at least short distance into laterally adjacent cells within the L1 layer of stage 3 flower buds, as shown by fluorescence recovery after photobleaching (FRAP) (Fig. 1). In view of these uniform results, it is tempting to speculate that the lateral MADS TF:GFP transport occurs by simple diffusion through predominantly primary PDs. These are laid down during cytokinesis in the division walls between the clonally related L1 cells, which maintain their layered SP L2

FD PP

PP

SP

L1

L3

Fig. 1 Schematic representation of the hypothetical movement of floral homeotic MADS box proteins based on research in A. thaliana and A. majus in an A. thaliana floral bud of a stage 4 to 5 (staging according to Smyth et al. (1990)), when sepal and petal primordia have already separated from the floral dome. Migration of AG from L2 to L3 is hypothetical as is lateral movement of all proteins in L2 and L3. AP3 does not migrate from L2 to L1; however, its A. majus ortholog DEF does. Also, acropetal movement of PI is hypothetical while its A. majus ortholog move in this direction. Black arrows indicate the direction of movement, AP3 is represented by green dots, PI by turquoise dots, AG by red and SEP3 by yellow dots. Layers 1, 2, and 3 abbreviated by L1, L2, and L3, respectively. SP sepal primordium, PP petal primordium, FD floral dome. Gray lines indicated the postulated barriers for diffusional transport between the organ primordia

A. Becker, K. Ehlers

organization through anticlinal divisions (Ehlers and Große Westerloh 2013). However, a general mechanism allowing PD transport of MADS proteins within their organ primordia can be refuted, since the MADS fusion proteins differed significantly in their capacities for basipetal transport between the L1 and L2 cell layer of inflorescence and stage 3 to 5 flower meristems (Urbanus et al. 2010). At this interface, the cells are mainly interconnected by secondary PDs which develop independently of cytokinesis in cell walls with massive postdivisional longitudinal expansion. Compared to primary PDs, transport through secondary PDs seems to be more selective, with restricted diffusional capacities, but higher competences for targeted transport (Burch-Smith et al. 2011; Ehlers and Große Westerloh 2013). Of the four MADS TF:GFP constructs under investigation, only AG:GFP moved basipetally into the underlying L2 cells when expressed from pML1, which suggests a specific, targeted PD transport mechanism (Urbanus et al. 2010). Moreover, the capacities for AG:GFP transport between layers appear to be cell-type or organ-type-specific, as the extent of transport was significantly higher in floral meristems than in inflorescence meristems and rosette leaves, where the native AG protein is absent. The hypothesis of AG acting as an NCATF for interlayer signaling during flower development is in accordance with the finding that pML1::AG:GFP is sufficient to rescue the ag mutant flower phenotype, while pML1::AP3:GFP only partially complements the ap3-3 mutant flower phenotype (Urbanus et al. 2010). Moreover, studies on genetic mosaic chimeras demonstrated that AG provides nonautonomous floral developmental information in the acropetal direction from the L2 to the L1 (Sieburth et al. 1998), whereas Jenik and Irish (2001) reported neither basipetal nor acropetal interlayer transport of AP3 (or PI) by immunolocalization in genetic mosaics and suggested that direct or indirect targets of AP3 rather than AP3 itself exert noncell autonomous developmental control (for PI, see Bouhidel and Irish 1996). However, the latter results are not fully consistent with the observations made for DEFICIENS (DEF) and GLOBOSA (GLO), the Antirrhinum majus orthologs of the B-class MADS TFs AP3 and PI from A. thaliana, which were identified as NCATFs by immunolocalization in Antirrhinum periclinal chimeras (Perbal et al. 1996). In contrast to AP3 in Arabidopsis, DEF and GLO in Antirrhinum moved acropetally into the mutant L1 and had moderate noncell autonomous B function effects on the mutant L1 phenotype, when the genes were only expressed in the L2 and L3 of floral buds. PD transport of DEF seems to be unipolar in the acropetal direction from L2/L3 into the L1, since basipetal PD transport or B function effects in the underlying mutant tissues were not observed after exclusive DEF expression in the L1 (GLO was not examined). This illustrates that PD transport

capacities of evolutionary conserved, orthologous floral homeotic genes, like DEF and AP3, have probably undergone distinct evolutionary specifications, leading to the diversification of their noncell autonomous function in distinct plant species (Han et al. 2014). Lateral PD transport of DEF and GLO in Anthirrhinum chimeras may be restricted at the boundaries of gene expression domains (Perbal et al. 1996), and similar conclusions can be drawn when details of the work of Urbanus et al. (2010) are reviewed. As might have been expected, the obligate heterodimerization partners AP3 and PI, both fused to GFP and constitutively expressed from pATML1, could not enter the nuclei in epidermal sectors of those floral bud whorls, in which the respective endogenous partner protein was missing, namely, PI in the first whorl and AP3 in whorl 1 and 4 (Urbanus et al. 2010). However, this finding also indicates that significant amounts of AP3, PI, and the heterodimers may be unable to move laterally within the L1 across the boundaries of gene expression between whorl 1 and 2 (and between whorl 3 and 4 for AP3 and the heterodimer). Interestingly, the same proteins are able to move between L1 cells within their expression domain as shown by FRAP in pATML1::AP3:GFP/pATML1:: PI:GFP double transgenic plants. It can be speculated that unspecific diffusional PD transport of floral homeotic proteins might be locally limited at the floral whorl boundaries, so that any symplastic exchange across these symplastic barriers must be targeted and strictly controlled to permit distinct floral organ identity and morphogenesis of distinct floral organs. A downregulation of diffusional symplastic exchange with the adjacent tissues is also a prerequisite for the development of highly specialized cell types like guard cells and root hairs, and it accompanies physiological or morphogenetic switches like the onset of dormancy or the transition to flowering (Burch-Smith et al. 2011; Ehlers and Große Westerloh 2013). Later in flower development, when ovules are formed, AG is expressed in the funiculus and the integuments, but the AG protein moves out of its expression domain and can be detected in the chalaza and nucellus (Urbanus et al. 2009). Here, it possibly acts together with SEP3 and BELL1 (BEL1) in the gradual restriction of WUS expression to the nucellus, which may confine the proper site of integument development from the chalaza (Brambilla et al. 2007; Urbanus et al. 2009). Other examples of incongruence between transcript presence and protein localization are the asymmetric distribution of SEP3 in the epidermis of developing petals or the presence of FRUITFULL (FUL) at the replum/valve margins of developing fruits (Urbanus et al. 2009). However, the biological relevance of these findings needs to be further examined. Taken together, we find several examples for noncell autonomous effects of TFs involved in floral organ initiation and morphogenesis, which point to highly controlled PD transport mechanisms that vary between species, cell and organ types,

Arabidopsis flower development review

and even between distinct interfaces of the same cells. The complexity of the regulatory network may be even higher, as a large number of TFs interact in a small tissue zone and only a few of them have been investigated so far with respect to their PD transport capacities. Thus, the overall concept of the role of PD transport in floral organ development remains rudimentary and needs more attention, while enormous progress has been made in the past years in understanding the noncell autonomous control mechanisms regulating flower induction or floral meristem formation, maintenance, and termination (Han et al. 2014; Holt et al. 2014; Vaddepalli et al. 2015).

Floral homeotic complex formation Most eukaryotic TFs require the physical interaction with at least one other protein of similar shape, in many cases of the same family to form a functional, DNA-binding complex (Amoutzias et al. 2008). The floral homeotic proteins, most of them members of the type II MADS domain family, are no exceptions to this rule, and thus, protein interaction analyses have been an integral part in the effort to elucidate the mechanisms required for flower development. Type II MADS box proteins are composed of four functionally distinct domains: the MADS domain (M) is located at the N-terminus required for DNA binding, C-terminal of the MADS domain is the intervening (I) domain, and following is the Keratin-like (K) domain. This domain includes two to three amphipathic helices and mediates dimeric protein interactions while the I domain is required for the specificity of dimeric protein interactions. The C-terminal (C) domain found at the C-terminus is variable in length and sequence (Riechmann et al. 1996; Yang et al. 2003). MADS box proteins generally recognize and bind to DNA target sequences with the consensus motif CC(A/T) 6 GG or CTA(A/T) 4 TAG termed CArG-boxes (reviewed in de Folter and Angenent 2006). For example, SEP3 proteins associate into dimers via a large hydrophobic interface provided by helix 1 of the K domain and salt bridges between amino acids in helix 2 (Puranik et al. 2014). However, the pattern of amino acids providing the hydrophobic interface is highly conserved between the SEP and the other floral homeotic proteins; yet, their dimerization partners differ drastically. It remains unknown which factors determine the dimerization specificity to explain the differences in protein interaction partners among floral homeotic MADS box proteins observed. Dimeric interactions of floral homeotic MADS box proteins It has been known for a long time that the B function proteins AP3 and PI act as an obligate heterodimer to bind to CArG boxes, as shown by multiple techniques, such as

immunoprecipitation (IP), yeast two-hybrid assays (Y2H), and bifluorescence complementation (BiFC) (Immink et al. 2009; Riechmann et al. 1996; Yang et al. 2003). While AP3PI heterodimers are able to bind to CArG-boxes, homodimers of the B function proteins or heterodimers of a B function protein with either AP1 or AG may form but are unable to bind to DNA (Riechmann et al. 1996). The protein interaction of AP3 and PI is also required for nuclear localization, as either protein alone is not able to enter the nucleus but together they may reconstitute a nuclear localization signal (McGonigle et al. 1996). This indicates that only specific protein complexes allow nuclear localization, CArG box binding, and thus floral homoetic complex activity. A large Y2H experiment comprising all A. thaliana MADS box proteins was conducted by de Folter et al. (2005) yielding large numbers of dimeric protein interactions between type II floral MADS box proteins. While both AP3 and PI are unable to form heterodimers with any other MADS domain protein as full-length proteins, the other floral MADS box proteins are rather promiscuous and interact with each other to some extent, and all of them interact with at least one SEP protein; AP1 even interacts with all four SEP proteins. This is especially interesting as the protein with highest sequence similarity to AP1, CAULIFLOWER (CAL), which has no role in specifying sepal identity but in floral meristem identity (Alvarez-Buylla et al. 2006), interacts with SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) only, a flowering time regulator (de Folter et al. 2005); AG interacts with the SEP1, SEP2, and SEP3; SHP1, SHP2, and STK all form heterodimers with SEP1 and SEP3 only (de Folter et al. 2005). The ovule identity proteins SHP1, SHP2, and STK interact with the SEP proteins, flowering time regulators such as SOC1 and AGAMOUS-LIKE24 (AGL24), and AGL15, an embryo development regulator (Thakare et al. 2008; Theißen and Saedler 2001; de Folter et al. 2005). Based on Y2H experiments, the SEP proteins interact differentially with other floral MADS box proteins when considering the number of interactors (de Folter et al. 2005). While SEP1 interacts with 17 MADS protein interaction partners, among them are AGL6, ARABIDOPSIS BSISTER (ABS), AP1, FUL, AG, SHP1, SHP2, and STK; SEP2 interacts with five only, among them is AG and ABS. SEP3 interacts with as many as 12 other MADS proteins, among them AGL6, ABS, AG, SHP1, SHP2, AP1, and FUL. SEP4, of which two splicing isoforms have been identified, interacts with three MADS box proteins only, including AP1 and FUL (de Folter et al. 2005). SEP1, SEP2, and SEP4 belong to one phylogenetic clade and SEP3 to another that originated by a duplication predating the angiosperm origin; their genetic redundancy seems astonishing (Ditta et al. 2004; Zahn et al. 2005). Not only the number of protein interaction partners, but also the ability for cooperative DNA binding differs between the SEP proteins (Jetha et al. 2014) supporting the idea that the SEP

A. Becker, K. Ehlers

Several multimeric complexes comprising only floral homeotic MADS box proteins have been documented already (Fig. 2a): PI-AP3-AG-SEP3 all need to be expressed in a Y2H experiment to activate reporter gene expression, with PI-AP3SEP3 and PI-AP3-AG interaction also documented by immunoprecipitation experiments and also PI-AP3-AP1 interaction. Interestingly, the overexpression of PI, AP3, and SEP3, all driven by constitutive promoters, leads to the conversion of cauline leaves into petal-like structures providing compelling evidence that these three proteins are sufficient for petal organ identity (Honma and Goto 2001). Electrophoretic mobility shift assays (EMSAs) have demonstrated that AP3-PI-SEP3SEP3 complexes are able to bind to two CArG boxes and that this tetramer forms preferentially over other tetramer combinations using these three proteins in vitro (Melzer and Theißen 2009). Trimeric AP3-PI-SEP3 complexes were also shown to form in planta (Immink et al. 2009), but their DNA binding and tetrameric complex formation have not been documented yet. Meanwhile, several other protein complexes including floral homeotic MADS box proteins have been identified, composed of only MADS box proteins or including proteins of other TF or transcription modulating families: AGL13, a protein related in sequence to the SEP proteins is able to replace SEP3 in an PI-AP3-AG-AGL13 complex and (AG-AGL13)2

proteins act largely but not fully redundantly. Interestingly, SEP3 has been termed the Bglue^ protein between mainly florally expressed MADS box proteins, but it is SEP1 that interacts with more MADS box proteins, and the same number of mainly florally expressed MADS-box proteins (Immink et al. 2009; Puranik et al. 2014; Kaufmann et al. 2009; Melzer et al. 2009). However, most of the interactions of florally expressed MADS box proteins have not been tested by independent methods like biochemical assays or BiFC (Miernyk and Thelen 2008; Bhat et al. 2006), and thus, the chances for false-negative and false-positive interactions are high. Multimeric interactions of floral homeotic MADS box proteins The orchestration of floral organ identity and regulation of floral organ morphogenesis in A. thaliana is achieved by MADS box proteins that not only assemble into homodimers and heterodimers, but which act mainly in multimeric complexes (Honma and Goto 2001; Theißen and Saedler 2001). In these complexes, MADS box protein dimers may associate with other MADS dimers, but also with other transcriptional activators or repressors and chromatin remodelers (Hsu et al. 2014; Pajoro et al. 2014; Brambilla et al. 2007; Melzer and Theißen 2009). Fig. 2 a Selection of important direct target genes of the MADS box protein complexes. At the top: protein complexes composed of only MADS box proteins; at the bottom: complexes including also other transcription factors or coregulators are depicted. The gray arrow symbolizes temporal occurrence and spatial arrangement of the floral organs in respect to the center of the flower. b Schematic representation of the pathways the individual floral homeotic proteins are regulating. Arrows indicate activation of genes or pathways, lines ending in bars indicate repression of gene expression or pathways

a

AP1, AP3, PI, SEP3

AP1, AP3, PI, SEP3, CUC, PTL, SUP, SPL, UFO

AP3, PI, AG, SEP3, CUC, PTL, SPL, SUP, UFO

PI AP3

PI AP3

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SEP AG 3

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AGL24, FD, TOE, SVP, SOC1

AGL24, FD, INO, SHP2, SOC, SVP, TOE1

sepal and early floral meristem

petal

SEU LUG

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AG, SEP3 SHP1

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carpel SEU

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b gibberellic acid homeostasis AP 1

organ patterning shoot program

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phytohormone response organ boundary specification stamen identity & development carpel identity & development

ovule

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phytohormone response carpel identity & development leaf program

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organ growth SEP 3

phytohormone response floral transition

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Arabidopsis flower development review

complexes can be identified by FRET analyses (Hsu et al. 2014). However, the range of dimeric interactions of AGL13 is limited to four proteins only, including SHP1 and AG as floral homeotic proteins only (de Folter et al. 2005) indicating that AGL13 may not be able to fully complement for SEP protein function. Protein complexes that may play a pivotal role in ovule development have been identified by yeast three-hybrid analysis. These consist of the homeodomain TF BEL1 and SEP1 or SEP3 plus either AG, STK, SHP1, or SHP2 (Brambilla et al. 2007) with different functions depending on the complex composition. While a SEP-AG complex promotes carpel identity, a STK-SHP-SEP complex inhibits carpel identity but promotes ovule identity. BEL1-AG-SEP limits WUS activity to the nucellus to ensure proper outgrowth of the ovule’s integuments (Brambilla et al. 2007). MADS box proteins not only interact directly with other TFs, but also with transcriptional coregulators and chromatin remodeling proteins thus providing a direct link to the transcription machinery (Fig. 2a). Earlier studies have shown already that protein interactions of AP1 and SEP3 with the pleiotropically acting transcriptional corepressors LEUNIG (LUG) and SEUSS (SEU) also play a role in flower development when repression of target genes is established, such as strong AG repression by AP1-LUG-SEU and SEP3-LUG-SEU in the first two floral whorls and weak AG repression in whorls 3 and 4 where AP1 is not expressed (Sridhar et al. 2006). Also, nucleosome-associated factors that bind to the floral homeotic proteins are numerous, among them are the histone demethylase RELATIVE OF EARLY FLOWERING6 and the chromatin remodelers BRAHMA, CHROMATIN REMODELING FACTOR17, and SPLA YED (Smaczniak et al. 2012). These results show that MADS box proteins not only form complexes among themselves but seem to be the core of large multimeric protein complexes that may direct the transcription of target genes directly via the incorporation of transcriptional repressors and by their capacity to directly act on chromatin structure and histone modifications. The C-terminal domain of MADS box proteins seems to be the determinant of ternary and quaternary complex formation, as several lines of evidence show. The C-terminal domain of SEP3 and SQUAMOSA, the AP1 homolog of Antirrhinum majus, mediates these complexes (Honma and Goto 2001; Egea-Cortines et al. 1999). It is also the C-terminus of AP1 and SEP3 that interacts with SEU (Sridhar et al. 2006), and the C-terminus of SEP3 that interacts with the homeodomain protein BEL1 (Brambilla et al. 2007). Work by Lange et al. (2013) shows that the short PI motif of PI-like proteins is required not only for the mediation but also for the selectivity of an E. californica protein complex consisting of PI-, SEP3-, and AG-like proteins. In Arabidopsis, this selectivity is most likely also relevant, but realized by a different, so far unknown mechanism, as the PI motif of the Arabidopsis PI protein is

mutated (Lange et al. 2013). While the K domain and Cterminal domain of MADS box proteins are required for mediating binary and multimeric interactions, respectively, it remains unclear, which amino acids mediate the specificity of ternary complex formation and how conserved the mechanism is. Another unresolved question is how other floral homeotic MADS box proteins are assembled in higher-order complexes, such as SEP1, SEP2, and SEP4. And, why are they able to act redundantly with SEP3 at the genetic level, but are unable to participate in interactions at the level of dimers and possibly also at higher-order complex formation?

Target genes of floral homeotic proteins The search for target genes of floral homeotic proteins reveals how the proteins regulate their own expression, how they influence expression of other floral homeotic genes, and which other direct targets they regulate. The latter one provides information on the developmental and biochemical pathways that generate the floral organs once organ identity is established, by directing three-dimensional patterning, directional growth, and tissue differentiation (Fig. 2a, b). Until only a few years ago, the identification of direct targets for floral homeotic genes was a very tedious process that required an inducible expression system with transcriptome analysis based on microarray hybridization and resulted in few candidates (Mara and Irish 2008). With the advent of chromatin immunoprecipitation (ChIP) coupled with high throughput sequencing techniques (ChIP-Seq), the number of putative target genes increased by magnitudes (Kaufmann et al. 2009, 2010; ÓMaoiléidigh et al. 2013; Mizzotti et al. 2014; Wuest et al. 2012). The floral homeotic A protein AP1 was the first one to be analyzed by ChIP-Seq and yielded well over 2000 high confidence target genes (Kaufmann et al. 2009). However, complementary transcriptome analysis by microarray hybridization observing early responses of induced AP1 transcription in ap1 cal mutants revealed that only around 10 % of the potential target genes are differentially expressed upon AP1 induction. This may be due to the fact that AP1 requires other proteins to bind to CArG boxes or that AP1 is only fine-tuning gene expression of many target genes. The observation of low differential expression among direct targets is not specific to AP1 but found throughout this type of analysis (Wuest et al. 2012; Kaufmann et al. 2009, 2010) Several genes known for their role in controlling AP1 expression such as the flowering time regulators FD and TARGET OR EAT1 (TOE1) are repressed by AP1, suggesting a role for AP1 in generally suppressing floral repressors and genes involved in shoot formation and maintenance (Kaufmann et al. 2009). A large number of genes that are targets of both AP1 and SEP3 play a role in floral organ

A. Becker, K. Ehlers

identity specification supporting the finding that AP1-SEP3 heterodimers activate gene expression at early stages of flower development. Another group of AP1 target genes points toward AP1 function after floral organ identity specification, in processes such as gibberellic acid homeostasis and floral organ patterning (Kaufmann et al. 2009). While we know that AP1 has dual functions as a repressor and activator of gene expression, we lack information on the protein composition of the repressing complex. As most activated genes are also targets of SEP3, it seems likely that the AP1-SEP3 dimer is involved in the activation of gene expression and less so in the repression of gene expression. Possible candidates to interact with AP1 in the protein complex required for repression of the vegetative development program are the MADS box proteins SOC1, SHORT VEGETATIVE PHASE (SVP), or AGL24, as these are able to dimerize with AP1 (de Folter et al. 2005) and act only until very early stages of flower development (Gregis et al. 2009). Dimers of AP1AGL24 and AP1-SVP associate with LUG and SEU to form a corepressor complex that binds to CArG boxes in the promoter region of AG, AP3, PI, and SEP3 to repress their expression. And, in these complexes, SOC1 is able to replace SVP and AGL24. And, while AP1-SEP3 containing protein complexes may promote expression of B and C class genes, AP1AGL24 and AP1-SVP associate with LUG and SEU to repress B, C, and E class gene expression (Gregis et al. 2009). The floral homeotic proteins AP3 and PI bind as obligate dimers to CArG boxes, and this finding is also corroborated by the identification of their binding sites, which largely overlap (Wuest et al. 2012). AP3 and PI bind to ~470 target genes that show differential expression in response to the presence of AP3 and PI. Many of these target genes are also targets of SEP3 and AP1, a finding that corroborates previous results showing that AP3-PI-SEP3 and AP1-SEP3 protein complexes exist in planta and further showing that they are functional in respect to controlling the expression of target genes (Honma and Goto 2001; de Folter et al. 2005). The AP3-PI dimer represses some genes and activates others, as was shown already for the AP1 containing protein complexes. The most important group among the genes directly repressed by AP3-PI are TFs involved in carpel and ovule development, such as SHP2 and the YABBY TFs CRABS CLAW (CRC) and INNER NO OUTER (INO) (Wuest et al. 2012). In the absence of AP3 or PI, CRC expression expands into the petal and stamen primordia; thus, AP3 and PI restrict CRC expression and possibly also expression of other genes to whorls 1 and 2 of the flower. And, while binding sites of AP3, PI, AP1, and SEP3 overlap in the promoter of SHP2 suggesting that an AP3-PI-SEP3-AP1 may bind to repress transcription, the putative binding sites of the CRC promoter of AP3, PI, and AP1, but not SEP3 overlap, possibly indicating that SEP3 is not involved in the CRC repressing complex (Wuest et al. 2012).

The AP3-PI dimer binds to and transcriptionally activates genes required for petal and stamen development, such as UNUSUAL FLORAL ORGANS (UFO), SPOROCYTELESS (SPL), and PETAL LOSS (PTL). Also, the organ boundary gene SUPERMAN (SUP) and genes involved in response and homeostasis of the hormones abscisic acid, auxin, gibberellin, and jasmonate are among the targets, indicating that a diverse array of processes, including extensive hormone signaling, orchestrates stamen and petal development (Wuest et al. 2012). The C function protein AG has been shown to act together with AP3, PI, and SEP3 in higher-order complexes to regulate gene expression (Honma and Goto 2001), implying that several targets of the AP3-PI dimer overlap with AG conferring stamen organ identity and stamen development. Among the common targets is AG itself, UFO, SUP, and CUP-SHAPED COTELYDONS (CUC), all required for stamen development or third whorl organ positioning (ÓMaoiléidigh et al. 2013). But, while stamen identity and development is controlled by the C function and B function together, carpel development is not. However, there are common targets of AG and AP3-PI in the carpel development pathway, such as CRC and SHP2, and while AG alone appears to activate their expression to initiate fourth whorl organ development, in conjunction with AP3-PI, AG is repressing their activity to allow third whorl organ development (ÓMaoiléidigh et al. 2013). Ever since the 18th century, it has been hypothesized that floral organs are modified leaves (Goethe 1790). And indeed, when B and C function genes are ectopically expressed, floral organ-like structures appear on leaves (Honma and Goto 2001), indicating that B and C function genes are either able to repress the leaf development program or that B and C function genes add parts of the floral development program on top of the leaf program. Work by ÓMaoiléidigh et al. (2013) shows that both scenarios seem to apply to at least AG activity. Among the genes activated by AG, those highly expressed in male and female gametophytic tissues are overrepresented, while regulators of leaf development are overrepresented among the genes repressed by AG early in flower development. This suggests that AG is able to act on both repressing leaf development and activating reproductive development. The E function protein SEP3 interacts with most florally expressed MADS box proteins (de Folter et al. 2005), implying that many target genes of the A, B, and C floral homeotic genes overlap with those of SEP3. ChIP experiments conducted by Kaufmann et al. (2009) aimed at identifying these direct targets of SEP3 and discriminated between targets specific for vegetative and others specific for reproductive development in the flower by using the ag mutant that lacks stamens and carpels. All floral homeotic genes, with the exception of only STK and CAL, exhibit SEP3 binding sites in the promoter, 5′ UTR, or introns of the coding regions. This suggests a high level of transcriptional autoregulation of the

Arabidopsis flower development review

floral MADS box proteins among themselves, which has been already shown for the AG-SEP3 dimer (Gomez-Mena et al. 2005), and is extended also to AP3 and PI, such that SEP3 strongly activates B function expression leading, in concert with AG, to the start of the floral developmental program. On the other hand, genes that regulate the transition from shoot apical meristem to floral meristem are repressed by SEP3 (Kaufmann et al. 2009). In addition to regulating florally expressed MADS box genes, other TF families are also among the targets of SEP3. Among these are the bHLH, TCP, GRAS, ARF, AUX-IAA, or Trihelix TFs, indicating that SEP3 is involved in the regulation of organ growth (TCP), and responses toward the phytohormones brassinosteroids (GRAS) and auxin (ARF, AUXIAA, and Trihelix). The activity of the ovule identity and seed development regulator STK commences comparatively late. When the carpel floral organ identity is already laid down, the gynoecium is initiated and STK is expressed when the carpel margins generate meristematic tissue from which the ovules arise. Transcriptome analyses of STK revealed direct and indirect targets and based on the late onset of STK activity, other MADS box genes are hardly among its targets, with the exception of ABS, a gene involved in endothelium development (Nesi et al. 2002; Mizzotti et al. 2014). Interestingly, a large fraction of genes differentially expressed between wild-type and stk mutants are related to metabolic processes. It was shown that STK represses the accumulation of proanthocyanidins (PA) in the inner seed coat layer by modifying chromatin marks at the BANYULS (BAN) promoter (Mizzotti et al. 2014). Unlike the targets of the MADS box proteins discussed earlier, which are predominantly other TFs, BAN is a metabolic enzyme required for anthocyanin biosynthesis and with this and several other PA biosynthesis genes differentially expressed, STK seems to regulate metabolic processes rather than development (Mizzotti et al. 2014). In summary, the analyses of direct targets of floral homeotic MADS box proteins allow some general conclusions as to how MADS box genes regulate floral organ identity as well as growth and differentiation of the floral organs. First, the floral homeotic A, B, C, and E proteins target mainly other TFs, among those are floral homeotic genes, suggesting autoregulatory action, and other TFs that are involved in growth, organ boundaries and position, hormone response, and gametophyte development (Fig. 2). Thus, floral homeotic genes (except for STK) are not involved directly into regulating metabolic processes, but rather into orchestrating downstream TFs that then directly or indirectly regulate enzyme activity. Second, floral homeotic genes may act as repressors or activators of expression, and their exact mode of operation may depend on the protein complex composition they are participating in. These complexes may contain not only DNA-binding TFs such as MADS box proteins, but also

chromatin remodelers and transcriptional corepressors to interact with RNA polymerase II (Fig. 2). Third, a large proportion of MADS box protein target sites identified in ChIP-Seq experiments are apparently bound by those, but this does not lead to significant change in gene expression when transcriptome analyses are carried out. Fourth, floral homeotic genes are required for specifying organ identity, but their regulatory action is also essential for further growth and differentiation of the floral organs, as their expression persists until the latest stages of flower development and their target genes additionally point toward a function of floral homeotic genes until the floral organs are matured. Many open questions regarding the molecular mechanisms governing flower development remain, and especially in respect to protein transport during floral organ initiation, there is a need to examine the biological relevance of noncell autonomous actions of TFs observed, since the findings obtained so far are mostly of descriptive nature. The molecular mechanisms controlling relevant symplastic transport events need to be elucidated then. How are the correct cargo proteins selected for targeted PD transport? Is there a specific PD targeting motif conserved among the MADS box proteins? And are there specific PD proteins which control the docking of individual TFs at particular cell interfaces? Moreover, we need a better picture of the dynamics of the PD networks throughout flower development to understand how developmental clues and NCATF transport are integrated. Which signals regulate the formation of primary and secondary PDs within and between cell layers in early flower buds and during growth of the floral organs? Are there developmental modifications of the PDs, e.g., to restrict diffusional transport at the whorl boundaries? And how are these modifications controlled? Finally, it should be considered that NCATF transport through PDs may interfere with TF protein complex formation and nuclear localization in competitive processes which would also exert spatiotemporal control on the noncell autonomous effects in the short-distance range. Future work has to unravel the protein complex components that account for the activation versus repression of target genes in neighboring whorls and why some bound DNA targets of MADS box proteins have an effect on transcription and other have not. And what target DNA property is important for MADS box dimer recognition? Moreover, identification of direct target genes of floral homeotic MADS box proteins has shown that depending on the protein complex they are part of, they may activate or repress a single’s gene expression, e.g., AG in combination with AP3 and PI represses CRC, but activates CRC when AP3 and PI are absent from the complex. There are many thousand CArG boxes which have been identified in the Arabidopsis genome that could be potentially bound by several MADS box proteins but yet they are not. And so far, we know very little about the molecular

A. Becker, K. Ehlers

mechanisms of transcriptional readout of MADS box protein binding. There is extensive cross talk of phytohormones and signaling involved in flower development, and many targets of floral homoetic genes are indeed genes involved in regulating hormone perception, homeostasis, transport, and biosynthesis. But how do phytohormones impact expression of floral homeotic genes on a molecular scale? Also, many processes downstream of floral homeotic targets genes are less well understood, such as how growth directions and cell proliferation rates change that ultimately result in the 3D-patterning of developing plant organs. And, how the detailed mechanistic insight gained from work in Arabidopsis can help understanding the nonfinite variations on the floral developmental program giving rise to the astonishing variation of floral form that evolution has created over the millennia. Acknowledgments We apologize to all authors whose papers we could not cite due to space constraints. Work in AB’s lab is funded by the German Research Foundation (DFG) (grants BE 2537/6-2, 8-1, 9-1, 121), and the Justus-Liebig-University Gießen, Germany.

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