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Katie Higgins and Jessica Kelly

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Patrick and Simone

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Tony Murray and Laura Leddy

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Sinead O'Boyle and Shannon Hill

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Presenter: Reem Buqrais Brendan Mc Donnell.

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Freddy Carew and Eabha Hussey

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Monday 15th October: Stacey Kerr and Asia Jordan

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Monday 15th October: Stacey Kerr and Asia Jordan

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-Jiaqi and Natalia

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Wednesday 10th- Caroline Dowling and Alannah Chalkley

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The SEPALLATA MADS-box protein SLMBP21 forms protein complexes with JOINTLESS and MACROCALYX as a transcription activator for development of the to... - PubMed - NCBI

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Functional conservation and diversification of class E floral homeotic genes in rice (Oryza sativa). - PubMed - NCBI

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Abstract

In rice panicle development, new meristems are generated sequentially in an organized manner and acquire their identity in a time- and position-dependent manner. In the panicle of the panicle phytomer2-1 ( pap2-1) mutant, the pattern of meristem initiation is disorganized and newly formed meristems show reduced competency to become spikelet meristems, resulting in the transformation of early arising spikelets into rachis branches. In addition, rudimentary glumes and sterile lemmas, the outermost organs of the spikelet, elongate into a leafy morphology. We propose that PAP2 is a positive regulator of spikelet meristem identity. Map-based cloning revealed that PAP2 encodes OsMADS34, a member of the SEPALLATA (SEP) subfamily of MADS-box proteins. PAP2/OsMADS34 belongs to the LOFSEP subgroup of MADS-box genes that show grass-specific diversification caused by gene duplication events. All five SEP subfamily genes in rice are expressed exclusively during panicle development, while their spatial and temporal expression patterns vary. PAP2 expression starts the earliest among the five SEP genes, and a low but significant level of PAP2 mRNA was detected in the inflorescence meristem, in branch meristems immediately after the transition, and in glume primordia, consistent with its role in the early development of spikelet formation. Our study provides new evidence supporting the hypothesis that the genes of the LOFSEP subgroup control developmental processes that are unique to grass species.

Introduction

The basic architecture of the inflorescence is determined by the identity and activity of meristems. Studies in the model eudicot species, mostly Arabidopsis, have allowed the basic model of the genetic control of flower initiation to be built (for a review, see Blázquez et al. 2006). LEAFY ( LFY) and APETALA1 ( AP1) are key regulators of floral meristem identity in Arabidopsis (Mandel et al. 1992, Weigel et al. 1992, Wagner et al. 1999). Other genes, such as CAULIFLOWER ( CAL) and SEPALLATA ( SEP), together with LFY and AP1, control floral meristem identity (Ferrándiz et al. 2000, Ditta et al. 2004). Once the floral meristem fate is established, the meristem starts to produce floral organs according to the mechanism known as the ABCE model (Goto et al. 2001).

Grass species show unique features of inflorescence development (Clark and Pohl 1969). A conspicuous characteristic of grass inflorescence is the spikelet, a small branch containing a variable number of flowers called florets. Because the spikelet is a terminal structure, the arrangement of spikelets rather than flowers is considered to define inflorescence architecture in grass species. A schematic of the rice inflorescence, called the panicle, is shown in Fig. 1A. During rice panicle formation, new meristems are sequentially generated. Newly formed meristems acquire the identity of a rachis branch meristem or terminate as a spikelet meristem, depending upon their position and the timing of their occurrence. The meristem that becomes a rachis branch meristem continues to generate next order lateral meristems that will eventually acquire the spikelet identity and terminate (Itoh et al. 2005). The key factor that determines the grass inflorescence form is the spatial and temporal regulation of spikelet meristem fate.

Because the spikelet is unique to grasses, unveiling the molecular and genetic control of spikelet initiation and development has been of great interest from both evolutionary and developmental points of view. So far, several genes that control panicle and spikelet development have been identified in grass species such as maize and rice (Bommert et al. 2005, Bortiri and Hake 2007). They can be classified based on the developmental steps they control. Genes of the first class, such as ABERRANT PANICLE ORGANIZATION1 ( APO1), RICE CENTROLADIALLIS ( RCN), GRAIN NUMBER1 ( GN1) and DENSE AND ERECT PANICLE1 ( DEP1), control the initiation of meristem identity (Ashikari et al. 2005, Ikeda et al. 2005, Ikeda et al. 2007, Huang et al. 2009, Ikeda et al. 2009). APO1, which encodes an F-box protein related to Arabidopsis UNUSUAL FLORAL ORGANS ( UFO), negatively regulates the spikelet meristem identity (Ikeda et al. 2005, Ikeda et al. 2007, Ikeda et al. 2009). In apo1 mutants, specification of the spikelet identity is accelerated, resulting in the formation of a small panicle. In contrast, an increased expression of APO1 caused the delay of spikelet identity determination, leading to an increased number of branches in the panicle. Constitutive expression of RCN, a TERMINAL FLOWER1 ( TFL1) ortholog, delays spikelet meristem identity and causes an extreme increase in the number and in the level of ramification of rachis branches, indicating that RCN can suppress the spikelet meristem identity (Nakagawa et al. 2002). GN1 and DEP1 were identified as quantitative trait loci that modulate grain yield through the control of the meristem phase transition to a spikelet meristem (Ashikari et al. 2005, Huang et al. 2009). GN1 encodes a cytokinin oxidase, an enzyme that catalyzes the degradation of cytokinins, implying an involvement of cytokinins in the control of spikelet meristem identity. Arrangement of spikelets in the panicle is disturbed in panicle phytomer1 ( pap1) mutants, suggesting that the PAP1 gene may play a role in the initiation of the spikelet meristems although its molecular nature remains to be determined (Takahashi et al. 1998).

Genes of the second class are required to maintain the spikelet meristem identity and/or to control the transition from spikelet to floret meristem identity. Mutations of these genes do not affect the arrangement of the spikelets but spikelet development is impaired. SUERNUMEROUS BRACT ( SNB), FRIZZY PANICLE ( FZP), LEAFY HULL STERILE1 ( LHS1) and EXTRA GLUME1 ( EG1) belong to this class (Jeon et al. 2000, Komatsu et al. 2003, Agrawal et al. 2005, Chen et al. 2006, Lee et al. 2006, Li et al. 2009). Finally, a third group of genes including SUPERWOMAN1, OsMADS3, OsMADS58 and DROOPING LEAF is required for floral organ development (Nagasawa et al., 2003, Yamaguchi et al. 2004, Yamaguchi et al. 2006).

Despite successful isolation and characterization of all the genes mentioned above, our understanding of the molecular basis of rice panicle development remains fragmented, and more molecular studies are needed for a full understanding of the process. In this study, we isolated a new mutant named panicle phytomer2 ( pap2) that shows abnormal panicle development including a disordered arrangement of spikelets, an increase of rachis branches and the elongation of rudimentary glumes and sterile lemmas. We show that PAP2 encodes OsMADS34, which belongs to the SEP subfamily of the MADS-box proteins. We suggest that PAP2 is one of the key regulators of spikelet meristem identity in rice.

Results

The panicle of the pap2-1 mutant produces more rachis branches

A wild-type panicle contains several primary rachis branches that are produced by the inflorescence meristem (IM). After producing several rachis branch meristems the IM loses its activity and is left as a vestige called a degenerate point at the base of the uppermost primary branch (Fig. 1A). The primary rachis branch contains a few secondary rachis branches at the bottom, several lateral spikelets and a terminal spikelet. Secondary rachis branches produce a few spikelets.

A recessive mutant with abnormal panicle morphology was identified through a screening of a rice population derived from tissue culture regenerated plants (Hirochika 2001). The morphological resemblance between the panicle of the mutant identified and that of a previously reported pap1 mutant (Takahashi et al. 1998) prompted us to test the genetic relationship between the two mutations. Because the genetic complementation test indicated that the mutation was not located in the PAP1 locus (data not shown), the mutant was named pap2-1. In the pap2-1 mutant, the arrangement of lateral organs, such as rachis branches and spikelets, was disordered and the panicle became irregularly denser, in sharp contrast to the well-defined phyllotaxy of the wild-type panicle ( Fig. 1B-D). Furthermore, agglomerates of a large number of branches and spikelets with arrested development were often observed ( Fig. 1 B, D, E).

The pap2-1 panicle appears smaller than that of the wild-type due to the suppressed elongation of nodes in the panicle ( Fig. 1B), but it contains more branches. We examined the branching pattern of pap2-1 in more detail. In this analysis, all spikelets were counted regardless of their development. The number of primary rachis branches in a rice panicle depends on the timing of IM deactivation. A significant increase in the number of primary rachis branches per panicle was observed in pap2-1, indicating that the IM activity was maintained longer in pap2-1 than in the wild-type ( Fig. 1F). The total number of lateral organs generated on primary rachis branches is slightly reduced in pap2-1 while it is significantly increased on the secondary branches ( Fig. 1G, H). In rice panicles, a few basal lateral organs on the rachis branches grow as next order branches and distal ones grow as spikelets. The shift from rachis branch formation to spikelet formation is delayed in pap2-1, resulting in the transformation of spikelets that arise early into rachis branches ( Fig. 1G, H). In the wild-type panicle, 31.0% of lateral organs produced in the primary rachis branches grow as secondary rachis branches, whereas 70.8% become secondary branches in pap2-1 ( Fig. 1G). This characteristic, namely production of more rachis branches, was again observed on the secondary rachis branches in pap2-1, leading to the formation of tertiary branches, which are not usually seen in the wild-type panicle ( Fig. 1H). The increase in the number of branches resulted in an increase in the total number of spikelets initiated per panicle ( Fig. 1I). Defects observed in pap2-1 panicles are schematically summarized in Fig. 1J. The panicle phenotypes observed in pap2-1 suggest that PAP2 positively controls spikelet meristem identity.

In contrast to the defects in the panicle, no alteration in the overall growth phenotype, including plant height, growth of tillers, the plastochron and the total number of leaves produced, was observed in pap2-1 during vegetative development ( Supplementary Fig. S1). Although flag leaves of the wild-type and pap2-1 emerged at the same time, the pap2-1 panicle appeared approximately 5 d later than that of the wild-type (82.6 ± 0.7 days in the wild-type, vs. 87.5 ± 0.7 d in pap2-1), most probably due to a delay in the elongation of the panicle node.

Abnormal morphology of the pap2-1 spikelet

The spikelet meristem produces lateral organs in a distichous phyllotaxy. The first set is two bracts called glumes. Then, one or more bracts known as lemmas are generated and a floral meristem initiates in the axil of the lemma. Because glumes in the rice spikelet are reduced in size, they are called rudimentary glumes. Inside of the rudimentary glumes are empty glumes and a floret containing a lemma, a palea, two lodicules, six stamens and a pistil (Yamaguchi and Hirano 2006). Only one floret is formed in a rice spikelet, but it is interpreted that a rice spikelet originally generated three florets and empty glumes are lemmas of two florets that have been lost during the course of evolution (Komatsu et al. 2003, Bommert et al. 2005). According to this interpretation, empty glumes would correspond to sterile lemmas in other grass species. Herein we use the term sterile lemmas instead of empty glumes to better compare the spikelet structure with that of other grass species.

The most conspicuous abnormality in pap2-1 spikelets is the elongation of the sterile lemmas ( Fig. 2A-F). Rudimentary glumes are also elongated, and an ectopic filamentous organ often develops in the axils of the rudimentary glumes, which are usually barren ( Fig. 2B, E, blue arrowhead). Approximately 60% of the spikelets in a pap2-1 panicle exhibited elongated rudimentary glumes, and 40% of them developed an ectopic filamentous organ ( Fig. 2G). Elongation of sterile lemmas was observed in all spikelets examined ( Fig. 2H). Approximately 13% of the sterile lemmas elongated to about half the size of the lemma, 65% grew to a similar size and 23% grew longer. In spite of the elongation, the appearance of the surface of the elongated sterile lemmas remained unchanged, indicating that their identity as sterile lemmas was maintained ( Fig. 2I, J). No detectable abnormality was observed in the floret ( Fig. 2K, L), including the morphology of the lemma and palea (data not shown). The abnormalities in pap2-1 spikelets are illustrated in Fig. 2 M.

PAP2 encodes a MADS-box transcription factor similar to SEPALLATA (SEP)

We isolated the PAP2 gene by map-based cloning. Fine mapping using 2,832 F ₂ plants allowed the PAP2 locus to be mapped to a 10,000 bp interval on chromosome 3, where only one gene, OsMADS34 (Os03g0753100), was predicted ( Fig. 3A). Sequence analysis revealed an insertion of Tos17, an endogenous retrotransposon, in the fourth exon of OsMADS34 in pap2-1 ( Fig. 3B). This insertion introduces a termination codon in the K-box of the protein ( Supplementary Fig. S2). The OsMADS34 mRNA accumulation was decreased to an undetectable level in pap2-1 ( Fig. 3C). These data suggest that PAP2 is OsMADS34 and that the defects in pap2-1 are most probably caused by the insertion of Tos17. We confirmed this hypothesis by introducing a wild-type genomic DNA fragment containing the OsMADS34 region into pap2-1 and achieving full complementation of the pap2-1 phenotype ( Fig. 3D). Finally, a reduction of OsMADS34 expression by RNA interference (RNAi) mimicked the pap2-1 phenotype ( Fig. 3E, F). Based on these results, we concluded that PAP2 is Os03g0753100, encoding a MADS-box protein, OsMADS34.

OsMADS34 is a member of the MADS-box gene family that regulates many aspects of plant growth and development (Malcomber and Kellogg 2005, Kater et al. 2006). PAP2/OsMADS34 is a type II MADS-box gene (there are >44 known type II MADS-box genes), a member of the SEPALLATA ( SEP) subfamily ( Fig. 3G). SEP genes are further divided into two major subgroups, namely the SEP3 and LOFSEP subgroups (Malcomber and Kellogg 2005, Zahn et al. 2005, Arora et al. 2007). Among the five genes of the SEP subfamily in rice, LHS1/OsMADS1, OsMADS5 and PAP2/OsMADS34 belong to the LOFSEP subgroup, and OsMADS7 and OsMADS8 are members of the SEP3 subgroup.

Expression pattern of the five rice SEP genes during early stages of panicle development

For a better understanding of the functions of the rice SEP genes in panicle development, the expression patterns of the five SEP genes during early stages of panicle development were examined ( Fig. 4). Although a comprehensive study of MADS-box gene expression of rice was reported previously, the earliest stage examined in the study was at the 0-3 cm panicle stage (Arora et al. 2007). Because all critical steps of spikelet and floral organ differentiation end before the panicle reaches 2 mm in size, and maturation and elongation of organs occur in the later stages, we decided to examine expression of the five SEP genes during much earlier stages of panicle development. We collected, under the microscope, panicles that were approximately 0.1-1 mm in length and further categorized them into five developmental stages as reported by Furutani et al. ( 2006). The expression of the five SEP genes started at various time during panicle development. A low level of PAP2/OsMADS34 mRNA accumulation could be observed from stage 1 when the primary rachis branches differentiate. The expression of PAP2/OsMADS34 continued through stages 2 and 3 and increased at stage 4 when spikelet organs differentiate. In contrast to the early onset of PAP2/OsMADS34, LHS1/OsMADS1 expression started from the spikelet meristem initiation stage (stage 4). High levels of LHS1/OsMADS1 expression were observed at the floral organ differentiation stage (stage 5). OsMADS5 showed a pattern of mRNA accumulation similar to that of PAP2/OsMADS34 but at relatively lower levels. Both OsMADS7 and OsMADS8 mRNAs are expressed predominantly at stage 5 as previously reported (Pelucchi et al. 2002).

Finally, we examined the spatial distribution of the expression of the three LOFSEP subgroup genes by in situ hybridization ( Fig. 5). PAP2/OsMADS34 mRNA accumulation was first observed in the IM and initiating primary rachis branches ( Fig. 5A, B), and continued in the lateral spikelet meristem and the floret meristem ( Fig. 5C, D). PAP2/OsMADS34 mRNA was also detected at the tips of the rudimentary glumes and sterile lemmas, as previously described by Pelucchi et al. ( 2002) ( Fig. 5E). LHS1/OsMADS1 expression was first observed in the entire region of the floret meristem ( Fig. 5H), and it started to be excluded from the meristem region as the lemma and palea primordial are formed, as previously reported ( Fig. 5I, J) (Prasad et al. 2001). OsMADS5 expression started slightly earlier than that of LHS1/OsMADS1 and was localized in the floret meristem ( Fig. 5L, M) and the palea primordia ( Fig. 5 N, O).

Discussion

PAP2 is a positive regulator of spikelet meristem identity in rice

Here we describe pap2, a new rice mutant that exhibits abnormal development of the panicle. In pap2-1, the early arising spikelet meristems are converted to rachis branch meristems, resulting in an increase in the number and the ramification level of rachis branches. Based on this observation, we propose that PAP2 is a spikelet meristem identity gene of rice. So far, only a few rice genes that control spikelet meristem identity have been reported. APO1 and GN1 work in the control of spikelet meristem identity, however, they suppress the spikelet's identity, an action opposite to that of PAP2 (Ashikari et al. 2005, Ikeda et al. 2005). On the other hand, DEP1 controls spikelet identity in a positive manner, as does PAP2 (Huang et al. 2009). RFL, a rice LFY ortholog, is another gene that negatively regulates spikelet identity (Rao et al. 2008). Elucidation of the genetic interactions among these genes is required in order to understand the mechanisms controlling rice panicle development. In addition to the conversion of spikelets to rachis branches, rudimentary glumes and sterile lemmas showed leafy morphology in pap2-1. This may be an indication of reduced floral fate, but we cannot rule out the possibility that PAP2 controls the identity of rudimentary glumes and sterile lemmas.

Among the spikelet meristem identity genes of rice, PAP2 is unique. pap2-1 is the only mutant in which disorganized positioning of branches and spikelets was observed in addition to the increase in the number of branches. In the other mutants, although the patterns of meristem identity are altered, the arrangement of lateral organs is well organized as in the wild-type panicle. Considering that all meristems in the rice panicle initiate as axillary meristems in the axils of bracts, the irregular pattern of branch formation in pap2-1 suggests that PAP2 may play a role in the control of phyllotaxy. This hypothesis should be examined in future studies.

Function of SEP subfamily MADS-box genes in rice panicle development

A large number of studies have shown that molecular mechanisms controlling the development of inner floral organs are relatively well conserved between grasses and eudicots (Kater et al. 2006, Yamaguchi and Hirano 2006). In contrast, little is known about the regulatory mechanisms that control the formation of outside organs that are unique to grass species and yet show a great variation among grass species. In this study, we show that PAP2/OsMADS34, belonging to the LOFSEP subgroup of the SEP family MADS-box genes, regulates the identity of the spikelet meristem and development of rudimentary glumes and sterile lemmas, the outermost organs in the spikelet. PAP2/OsMADS34 branches out with a group of monocot genes on a phylogenetic tree ( Fig. 3E). Phylogenetic analyses have shown that duplications of LOFSEP genes occurred near the origin of grasses to produce PAP2/OsMADS34, LHS1/OsMADS1 and OsMADS5 clades (Malcomber and Kellogg 2005, Zahn et al. 2005). The early diversification of monocot LOFSEP genes and their distinct expression patterns suggests that they might have contributed to generate diversity in the reproductive development among monocots. Notably, PAP2/OsMADS34 is required for spikelet meristem identity while LHS1/OsMADS1 is essential for normal development of the lemma and palea. Lemma- and palea-like organs are reiteratively generated in lhs1 loss-of-function mutants, and glumes are transformed into lemma- and palea-like organs by constitutive expression of LHS1/OsMADS1 (Prasad et al. 2001, Agrawal et al. 2005, Chen et al. 2006). There is a clear distinction between the expression patterns of PAP2/OsMADS34 and LHS1/OsMADS1 (Prasad et al. 2001, Arora et al. 2007, this study). Based on these observations, we postulate that grass-specific LOFSEP genes sequentially control spikelet development, in particular leafy organs in the spikelet. First, PAP2/OsMADS34 works to initiate spikelet meristem identity and to control the development of rudimentary glumes and sterile lemmas. Then, LHS1/OsMADS1 acts to maintain the spikelet meristem identity and controls palea/lemma organ identity, which form inside of the rudimentary glumes and sterile lemmas. No obvious defect was observed in the loss-of-function mutants of OsMADS5, the other member of the LOFSEP subgroup (Agrawal et al. 2005). High sequence similarity between OsMADS1 and OsMADS5 suggests that they may have redundant functions. Although loss-of-function phenotypes of OsMADS7 and OsMADS8, members of the SEP3 subgroup in the SEP subfamily, are yet to be described, the spatial distribution of their mRNA suggests that they probably control the identity of the inner three floral organs in a similar fashion to the Arabidopsis SEP3 subgroup genes (Pelucchi et al. 2002, this study). The five rice MADS-box genes are expressed sequentially from the initiation of spikelet meristem to floral organ development, so that they may control the entire process of spikelet development. This is in contrast to the four SEP genes of Arabidopsis, which redundantly act to regulate floral meristem identity and organ identities of all four whorls (Pelaz et al. 2000, Ditta et al. 2004).

Previous analysis of LHS1/OsMADS1 mRNA expression patterns and spikelet development in grass species has suggested that changes in the expression pattern of LHS1/OsMADS1 contributed to the diversification of spikelets (Malcomber and Kellogg 2004, Reinheimer et al. 2006). Our study also supports the idea that the LOFSEP subgroup of SEP genes, a class of genes specific to grasses, plays a crucial role in the control of spikelets that are unique to grasses. Analysis of the function of PAP2 orthologs in other grasses will facilitate our understanding of the molecular basis of the evolution of spikelet development.

Molecular network controlling rice spikelet development

SEP proteins function as transcription factors in the form of a tetrameric complex (Theißen 2001, Kaufmann et al. 2005). Specific functions of SEP genes depend on the combination of MADS-box proteins in the complex. For instance, the combination of class-E SEP and class-A AP1 determines sepal formation in whorl 1 (Pelaz et al. 2001). Similarly, rice SEP proteins could form a tetrameric complex to regulate spikelet and floret development. Indeed, interactions between LHS1/OsMADS1 and RAP1A/OsMADS15 or RAP1B/OsMADS14, both orthologs of Arabidopsis AP1, were reported (Lim et al. 2000). The significance of an AP1 and LHS1 combination for the determination of glumes and florets in grasses has been demonstrated (Preston and Kellogg 2007). Identifying partners of PAP2 in the transcription factor complex will be a prerequisite for further elucidation of the role of PAP2. Moreover, isolation of the PAP1 gene and determination of genetic and molecular interactions between PAP1 and PAP2 will lead to a more comprehensive understanding of rice spikelet development.

Materials and methods

Plant materials

Plants grown in a glasshouse under natural conditions were used for the analysis of pap2-1 phenotypes. For mRNA extraction and in situ hybridization analysis, plants (cv. Nipponbare) were grown in a growth chamber under short day conditions (12 h light 28°C, 12 h dark 24°C).

Scanning electron microscope analysis

Spikelets in wild-type (cv. Nipponbare) and pap2-1 were fixed in 2.5% glutaraldehyde overnight at 4°C, dehydrated in a series of ethanol solutions and substituted with 3-methylbuthyl acetate. Subsequently, samples were dried at critical point, sputter-coated with platinum and observed under a scanning electron microscope (S-4000; Hitachi, Japan) at an accelerating voltage of 10 kV.

Molecular cloning of the PAP2 gene

Rough mapping of the PAP2 locus was performed with simple sequence repeat (SSR) markers using the mutant F ₂ plants obtained from a cross between pap2-1 and wild-type (cv. Kasalath) plants. For fine mapping, cleaved amplified polymorphic sequence (CAPS) markers were generated based on single nucleotide polymorphisms identified in nucleotide sequences between Nipponbare and Kasalath.

For the complementation test, a genomic fragment of 6,420 bp containing the OsMADS34 gene was generated by BglII and EcoRV digestions from a bacterial artificial chromosome (BAC) clone OSJNBa0047E24, and subcloned into pBluescript to generate pBSMADS34. A 1,330 bp fragment containing the promoter region of OsMADS34 was amplified by PCR using TArget Clone-Plus- (TOYOBO, Japan) polymerase with the following set of primers: 5′-CACCATTGCAGCTACAGTA CACCC-3′ and 5′-CTTGTTCTCGATCCGCTGAA-3′. Another genomic fragment of 2,504 bp containing the OsMADS34 3′-untranslated region and a terminator was generated by HindIII digestion of BAC clone OSJNBa0047E24, and cloned into pBluescript to generate pBSMADS34Term. The amplified fragment of the OsMADS34 promoter was cloned into pBSMADS34Term to generate pBSPMADS34. The fragment containing the whole region of the OsMADS34 gene including the promoter, the coding region and the terminator was cloned into the binary vector pPZP (Fuse et al. 2001).

Suppression of OsMADS34 expression by RNAi

To suppress the expression of OsMADS34, a part of the coding region amplified with the set of primers 5′-CACCTTATGCTTCG CAAGATGCTG-3′ and 5′-AGGTCGCAGAGTTCATCAAG-3′ was cloned into pANDA (Miki and Shimamoto 2004, Miki et al. 2005) and transferred into wild-type rice (cv. Nipponabare).

Real-time reverse transcription-PCR (RT-PCR) analysis

Total RNA was extracted from very young panicles by using a Plant RNA Isolation Mini Kit (Agilent) according to the manufacture's protocol. After the DNase I treatment, the first-strand cDNA was synthesized using SuperScript III Reverse Transcriptase (Invitrogen, USA). RT-PCR was performed using the following primer sets: PAP2/OsMADS34, 5′-TTGATGAA CTCTGCGACCTAAA-3′ and 5′-TGCTGCAGTTTCCGTTCC-3′; LHS1/OsMADS1, 5′-GTGACCATTCCCTGCAGATT-3′ and 5′-GTCTGCTGCTTCATTGCTCA-3′; OsMADS5, 5′-TTGCAACT ACAACCTTAACTCATGT-3′ and 5′-GCAGCATTCATCAATC AAACC-3′; OsMADS7, 5′-GTGGCAACGGATTCTTCC-3′ and 5′-CGCATGAGTTGTTCATCTGC-3′; OsMADS8, 5′-ACGGAGC TTCAGAGAAAGGA-3′ and 5′-CCTGCTCCCACACTTGCT-3′; and ACTIN, 5′-GCCGTCCTCTCTCTGTATGC-3′ and 5′-GG GGACAGTGTGGCTGAC-3′. The relative abundances of mRNAs were measured with a LightCycler 480 system (Roche Applied Science, Germany) using ACTIN gene expression as a reference for normalization.

In situ hybridizations

In situ hybridizations were carried out as previously described (Kyozuka et al. 1998). To synthesize a digoxigenin-labeled antisense probe of PAP2/OsMADS34, a fragment containing the C-terminal region was amplified by PCR, cloned into pGEMT-easy vector (Promega, USA) and used as the template. The primer set used to amplify PAP2/OsMADS34 was 5′-GTAGA GGCAGCTCCCCCAC-3′ and 5′-GCTAGGCCATCCACTCAGG AGG-3′. Digoxigenin-labeled antisense probes of LHS1/OsMADS1 and OsMADS5 were synthesized by using full-length cDNAs, AK070981 and AK064184, respectively, as templates.

Funding

This work was supported by the Ministry of Agriculture, Forestry and Fisheries [Genomics for Agricultural Innovation, IPG-0001 to J.K.]; Ministry of Education, Culture, Sports, Science and Technologies [Grant-in-Aid for Scientific Research on Priority Areas to J.K.]; Ministry of Education, Culture, Sports, Science and Technology, Japan [Grant in-Aid for Scientific Research for Plant Graduate Student from Nara Institute Science and Technology to K.K].

Abbreviations

References

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5) which permits unrestricted non-commercial use distribution, and reproduction in any medium, provided the original work is properly cited.

# data[].attributes.attachment_link.title
PANICLE PHYTOMER2 ( PAP2 ), encoding a SEPALLATA subfamily MADS-box protein, positively controls spikelet meristem identity in rice | Plant and Cell Physiology | Oxford Academic

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In rice panicle development, new meristems are generated sequentially in an organized manner and acquire their identity in a time- and position-dependent manner. In the panicle of the panicle phytomer2-1 ( pap2-1) mutant, the pattern of meristem initiation is disorganized and newly formed meristems show reduced competency to become spikelet meristems, resulting in the transformation of early arising spikelets into rachis branches.

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Presenters:

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Background

Development of eukaryotic organisms is controlled by transcription factors that trigger specific and global changes in gene expression programs. In plants, MADS-domain transcription factors act as master regulators of developmental switches and organ specification. However, the mechanisms by which these factors dynamically regulate the expression of their target genes at different developmental stages are still poorly understood.

Results

We characterized the relationship of chromatin accessibility, gene expression, and DNA binding of two MADS-domain proteins at different stages of Arabidopsis flower development. Dynamic changes in APETALA1 and SEPALLATA3 DNA binding correlated with changes in gene expression, and many of the target genes could be associated with the developmental stage in which they are transcriptionally controlled. We also observe dynamic changes in chromatin accessibility during flower development. Remarkably, DNA binding of APETALA1 and SEPALLATA3 is largely independent of the accessibility status of their binding regions and it can precede increases in DNA accessibility. These results suggest that APETALA1 and SEPALLATA3 may modulate chromatin accessibility, thereby facilitating access of other transcriptional regulators to their target genes.

Conclusions

Our findings indicate that different homeotic factors regulate partly overlapping, yet also distinctive sets of target genes in a partly stage-specific fashion. By combining the information from DNA-binding and gene expression data, we are able to propose models of stage-specific regulatory interactions, thereby addressing dynamics of regulatory networks throughout flower development. Furthermore, MADS-domain TFs may regulate gene expression by alternative strategies, one of which is modulation of chromatin accessibility.

Flower Development

Floral Organ

Floral Meristem

Chromatin Accessibility

Growth Regulate Factor

Stem cells residing in meristems enable plants to produce new organs throughout their lives. Vegetative meristems in the shoot apex produce leaves, while reproductive meristems produce flowers or floral organs. The identities of different types of floral organs (sepals, petals, stamens, and carpels) are established by homeotic MADS-domain transcription factors (TFs) via modification of the leaf developmental programme []. Homeotic genes become activated in floral meristems through regulators that specify floral meristem identity. An important regulator of floral meristem identity in Arabidopsis is the MADS-box gene APETALA1 ( AP1), which has an additional role as homeotic regulator of sepal and petal identity []. Homeotic proteins specify different floral organ identities in a combinatorial fashion, mediated by protein interactions and formation of heteromeric quaternary protein complexes [-]. Homeotic genes can also enhance or repress each other's expression, resulting in a complex transcriptional regulatory network. Mediators of higher-order complex formation are the largely redundantly acting members of the SEPALLATA MADS-domain subfamily, SEPALLATA 1 to 4 (SEP1-4) [, , ]. Therefore, these proteins have an important role in the specification of floral organ identities. Members of the MADS-domain TF family also act in many other developmental processes in plants, regulating directly and indirectly the expression of thousands of genes in the genome (for review, see [, ]). Floral MADS-domain TFs are found in larger protein complexes together with chromatin remodeling and modifying proteins, as well as with general transcriptional co-regulators [, ]. These interactions are important for the regulation of gene expression by the MADS-domain factors [, , ]. The expression of floral homeotic MADS-box genes is also regulated at the level of chromatin structure: outside the flower and at the earliest stages of floral meristem development, these genes are repressed by Polycomb group (PcG) protein complexes that act in concert with earlier acting MADS-domain TFs and other transcriptional regulators []. The physical and genetic interactions between MADS-domain proteins and chromatin regulatory factors suggest an important role of these TFs in controlling chromatin dynamics during plant development. To gain a genome-wide perspective on the developmental dynamics of gene regulation in plants, we studied MADS-domain TF occupancy, chromatin accessibility, and gene expression changes at different stages of Arabidopsis flower development. Our findings suggest that MADS-domain TFs may induce changes in chromatin accessibility, and thereby they are able to set appropriate chromatin landscapes for following regulatory processes leading to meristem and organ differentiation during flower development. By combining DNA-binding data and expression data, we established stage-specific gene regulatory interactions in floral morphogenesis.

Developmental dynamics of floral gene regulation

We studied global changes in chromatin accessibility, gene expression, and DNA binding of two MADS-domain TFs at different stages of flower development (Figure ). To obtain sufficient stage-specific plant material, we used an inducible system for synchronized flower development based on a chemically inducible version of the AP1 TF expressed under the control of its own promoter in the ap1 cal mutant background ( pAP1:AP1-GR ap1 cal line). We analyzed different floral stages during which floral meristem specification (days 0 to 2), floral organ specification (day 4), and floral organ differentiation (day 8) take place []. In order to study chromatin accessibility at these different stages, we made use of DNase-seq []. Furthermore, we performed ChIP-seq experiments to identify stage-specific DNA-binding sites (BSs) of the two MADS-domain TFs, AP1 and SEP3. SEP3 is a direct target gene of AP1 and becomes strongly expressed around floral stage 3, when the sepal primordia arise (day 3 after floral initiation) []. Genome-wide expression analyses were performed in order to detect changes in gene activity between different floral stages.

The ChIP-seq experiments generated high confidence sets of TF-bound regions for each factor and time point (see Additional file : Table S1 for an overview and a list of TF-bound regions). Many TF binding events were common to the different time points (Figure A), this result also holds when we analyzed each biological ChIP-seq replicate independently (see Additional file : Figure S1). For example, 67% of AP1 target genes and 90% of SEP3 target genes identified at day 4 are also present in the day 8 dataset (see Additional file : Figure S2A). We also observed a number of stage-specific binding events and potential direct target genes, for example 21% of putative AP1 target genes at day 4 were not found at any other time point (see Additional file : Figure S2A). DNA binding of TFs is not a none-or-all phenomenon; rather, quantitative differences in TF occupancy can influence transcriptional behavior []. Therefore, we studied quantitative changes in AP1 and SEP3 binding levels between different time points [, ]. By comparing ChIP-seq peak scores as a measure of relative binding levels, we identified several hundred genomic regions with changes in TF occupancy (fold-change (FC) ≥2; Figure B and see Additional file : Table S2). In case of significant AP1 binding sites, 26% show differences between days 2 and 4, and 42% between days 4 and 8. For SEP3 binding levels, 1,118 (17%) genomic regions showed changes between days 2 and 4 and 1,003 (12%) between days 4 and 8 (Figure B).

To investigate whether differences in AP1 and SEP3 binding are associated with stage-specific functions of these TFs, we analyzed the over-representation of GO categories in the different datasets. GO enrichment analysis revealed that genes involved in pattern formation, meristem maintenance, organ formation, and polarity are mostly bound by AP1 and SEP3 at early developmental stages (see Additional file : Figure S2B). For example, STERILE APETALA ( SAP), a regulator of floral organ patterning [], and FILAMENTOUS FLOWER ( FIL) [] and ASYMMETRIC LEAVES 1 and 2 [], genes controlling axis specification, are among those genes. On the other hand, genes involved in hormonal signaling are more strongly bound at later developmental stages (see Additional file : Figure S2B). The results of stage-specific ChIP-seq experiments, in combination with gene expression data, therefore allow to identify stage-specific regulatory interactions.

Among the potential direct target genes of AP1 and SEP3, there is over-representation of specific TF families, and the degree of over-representation for a given family may vary between time points (see Additional file : Table S3), suggesting stage-specific regulatory interactions. A family that is over-represented among both AP1 and SEP3 targets at 2, 4, and 8 days ( P value <0.05) is the GROWTH REGULATING FACTOR (GRF) family (see Additional file : Table S3). In particular, all nine GRF family genes are significantly bound by SEP3 (FDR <0.001), although a quantitative difference in binding levels was observed, and five of them are bound by AP1 (Figure C). GRF genes have well-known roles in leaf growth [], but no known function in the determination of flower organ identity. Seven out of the nine Arabidopsis GRF genes ( GRF1, 2, 3, 4, 7, 8, and 9) contain a target site for miR396 [, ]. The floral phenotypes of plants overexpressing miR396a from the 35S or p ANT promoters largely resemble the phenotype of a weak ap1 mutant allele, ap1- 3, suggesting a role of these genes downstream of AP1. In ap1- 3 flowers, as well as in miR396a overexpression lines (Figure D), the second floral whorl is often occupied by petal-stamen mosaic structures [, ]. Plants overexpressing miR396a show also a reduction in carpel number (Figure D). Severity of the mutant phenotype directly correlates with the level of reduction in GRF transcript abundance (Figure D and see Additional file : Figure S2C). In summary, these results indicate that, apparently redundant GRF family members are regulated in different ways, and that the phenotype that was observed in the miRNA-directed knockdown lines probably reflects the combined function of these family members in floral meristem patterning and in floral organ differentiation.

We next investigated the relationship at genome-wide level between changes of MADS-domain TF binding and changes in the expression of closely adjacent genes (that is, genes with a binding site within a region 1 kb upstream of the start of the gene or inside the gene) (Figure E). We observed a correlation between changes in binding and changes in expression. Genes located near regions with decreasing TF binding preferentially showed a reduction in their expression level, whereas increased TF binding was associated with an increase in the expression of nearby genes (Figure E).

In summary, AP1 and SEP3 binding sites overlap substantially between time points, but there is also an important number of BSs specific for each TF at each time point. Moreover, we observed that dynamic changes in AP1 and SEP3 DNA-binding correlate with changes in gene expression.

Overlap and differences between AP1 and SEP3 DNA binding and potential direct target genes

We found a significant overlap for AP1 and SEP3 target genes (Figure A and see Additional file : Figure S3A), which is in agreement with previous observations that were made using different plant materials, antibodies for the AP1-GR fusion protein, and time points []. In agreement with the fact that SEP3 and AP1 form higher-order protein complexes with the B-class homeotic proteins APETALA3 (AP3) and PISTILLATA (PI), we observed a clear overlap between sets of potential direct target genes (see Additional file : Figure S3B) [].

Results from Drosophila have shown that while many TFs have common binding sites in the genome, quantitative differences in binding levels correlate with the specific biological functions of different factors []. Quantitative comparison of genomic regions that are bound by both AP1 and SEP3 at the same time point shows that between 70% and 80% of the regions have peaks of similar height for both TFs (see Additional file : Table S2). Nevertheless, depending on the time point, from about 8% to 2% of all bound regions are preferentially bound by AP1 while a higher number of regions are more strongly occupied by SEP3 (FC ≥2; Figure B). For example, SHN1, a regulator of epidermal cell morphology of floral organs [], is preferentially bound by AP1 at day 4. In contrast, CRABS CLAW ( CRC), which is involved in specifying abaxial cell fate in carpels and in nectary formation [], and TGACG (TGA) MOTIF-BINDING PROTEIN 9 ( TGA9), which is involved in anther formation [] are preferentially bound by SEP3 (Figure C). These genes are significantly upregulated throughout all stages of flower development in the gene expression microarray data (see Additional file : Table S4). We confirmed the microarray results by quantitative PCR (qPCR) (see Additional file : Figure S3C). Thus, differences in quantitative levels of TF occupancy may help to explain target-gene specificity of floral homeotic protein complexes.

Dynamics of chromatin accessibility during flower development

Mapping of DNase I hypersensitive sites (DHSs) is a well-established method to identify the location of active gene regulatory elements []. The DNase I enzyme preferentially digests DNA in regions of low nucleosome occupancy, and DNase I digest of chromatin followed by deep sequencing identifies open or accessible genomic regions at genome-wide scale. DHSs have been found to be correlated with genomic regulatory features such as transcription start sites (TSSs), enhancers, and TF binding sites []. Focusing on genomic regions nearby genes (3 kb upstream of the start of the gene and 1 kb downstream of the end of the gene), we found that the overall number of detected high-confidence (FDR <0.01) DHSs at the different time points after AP1 induction vary between 5,680 and 8,789 (see Additional file : Table S5). We observed a high overlap (98.7%) between the DHSs identified at day 8 compared with the 41,193 previously identified DHSs in wild-type inflorescences (stages 1 to 11) [], the larger number of DHSs in wild-type inflorescences may be a consequence of using tissue that represents a mixture of different stages. Whereas the majority of DHSs were invariant across consecutive time points (FC < √2), 1,370 quantitative changes in chromatin accessibility (measured as changes in DHS peak score) were detected. While there were only a small number of changes in DHS peak score comparing the different meristematic stages, the transition to organ differentiation (day 4 to 8) was found to be associated with the most changes in chromatin accessibility. There were significantly more differences between day 4 to 8 than between the earlier time points ( P <2.2e-16, χ ² test) (see Additional file : Table S6). A total of 1,304 DHSs (11.8% of all DHSs detected at days 4 and 8) show quantitative differences in DHS peak score between days 4 and 8, with a slight preponderance of changes leading to increased accessibility (Figure A). Distinct clusters of differentially expressed genes were identified: those specific to early meristematic stages (clusters 1 and 2), transiently activated (cluster 6) or repressed (cluster 4) and genes that are specific to later floral stages (cluster 5) (Figure B). The trends in gene expression are reflected in concordant changes in chromatin accessibility: for example, genes that are expressed predominantly during meristematic stages of flower development (cluster 2), show over-representation of decreasing DHSs towards later stages (day 8). On the other hand, genes that are specifically activated later during floral organ development (cluster 5) show preferentially concordant increase in accessibility (see Additional file : Figure S4A). These data support the idea that changes in accessible genomic regions are linked with different sets of genes being active in meristematic cells versus differentiating tissues.

Next, we studied the relationship between changes in accessibility level of AP1- or SEP3-bound regions and expression of closely adjacent genes. Change in chromatin accessibility between meristematic tissues and differentiating floral organs is related with a corresponding change of expression of nearby genes (Figure C). This relation is statistically significant for both AP1- and SEP3-bound loci comparing days 2 to 4 and days 4 to 8 ( P <0.001; χ2 test), where the proportions of upregulated genes are larger for regions with increased accessibility, and the proportions of downregulated genes are correspondingly smaller. Using members of the GRF family as an example, we analyzed how variations in chromatin accessibility were associated with differences in spatiotemporal gene activity. GRF8 shows an increased SEP3 BS between days 4 and 8 and GRF8 chromatin becomes more accessible in differentiating floral organs (day 8) (see Additional file : Table S6). GFP reporter gene analyses show that the GRF8 protein is, in contrast to other factors such as GRF2 and 5, not expressed in flower meristems, and its expression increases in differentiating organs (Figure D,E).

General meristematic regulators are found among genes with a decrease in both accessibility and expression, such as SHOOT MERISTEMLESS ( STM) (Figure E). These data are consistent with previous findings, which report STM expression mainly in meristems, while the expression is later restricted to cells in the gynoecium, which give rise to ovules []. A decrease in chromatin accessibility and expression is also found for loci that control early patterning processes in floral meristems, such as AINTEGUMENTA-LIKE 6 ( AIL6), CAULIFLOWER ( CAL) (Figure E), and STERILE APETALA ( SAP). These data are corroborated by previous studies that reported predominant expression of AIL6[], SAP[], and CAL[] in meristems and young developing floral organ primordia (see Additional file : Table S6 and see Additional file : Table S4). Among the genes that show an increase in accessibility during flower development are a number of genes with specific roles in floral organ development, as well as more general regulators of organogenesis and growth. For example, the SEPALLATA3 locus is among the earliest genes with increased accessibility (day 2). Other examples for genes with increased accessibility at day 4 include patterning genes like PHAVOLUTA ( PHV) (Figure E). All these genes show a corresponding increase in expression. Among the genes that show predominantly increased accessibility from day 4 to day 8 (see Additional file : Table S6) are for example TFs known to be involved in the formation of carpels, ovules and seeds, like ALCATRAZ ( ALC) and NGATHA3 ( NGA3) (Figure E and see Additional file : Table S6). In accordance with the idea that different promoter elements may control different aspects of gene regulation, we found that at a subset of those loci, individual DHSs change in opposite fashion: some DHS peaks increase, while others in the same promoter decrease (see Additional file : Figure S4B).

In summary, we found that changes in chromatin accessibility occur mainly between days 4 and 8 and that they correlate with changes in gene expression.

Footprints of MADS-domain TF binding sites in flower development

The binding of a TF protects the DNA from DNase I digestion, creating a specific 'footprint' []. We analyzed footprint patterns caused by protection of DNA upon AP1 or SEP3 binding. The time-series ChIP-seq data indicate that AP1 and SEP3 show quantitative differences in TF occupancy levels at different developmental stages (Figure ). As MADS-domain TFs assemble into protein complexes in a combinatorial fashion, these differences may reflect changes in complex composition resulting in changes in DNA-binding specificity. In line with previous results [, ], de novo identification of DNA sequence motifs in genomic regions bound by AP1 and SEP3 resulted mainly in motifs representing CArG boxes (see Additional file : Figure S4C). The generic CArG-box motif (hereafter named 'CArG box 1'), which was identified both in the AP1 and the SEP3 datasets, possesses [A/T] stretches of variable length outside the central CC[A/T] ₆GG core. Thus, for AP1 and SEP3 we identify a longer consensus sequence than the canonical CArG-box motif: TTxCC[A/T] ₆ GGxAA. A second CArG motif, lacking an [A/T] stretch on one side of the CArG-box, was identified in SEP3-bound regions (hereafter named 'CArG box 2'). The generic CArG-box 1 has a footprint with a central dip corresponding to the region that is highly protected to the cutting of DNase I, indicating a possible contact between the protein and the nucleotide at that position (Figure A). In contrast, CArG-box 2 shows a footprint that suggests exposure of the DNA in the centre of the CArG-box (Figure B). By comparing the frequency of footprints at different developmental stages (Figure , left panels), we found that the genomic sequences corresponding to CArG-box 1 are similarly bound at all developmental stages. In contrast, those corresponding to CArG-box 2 show increasing frequencies of footprints at day 8 compared to earlier time points. This suggests that CArG-box 2 is more predominantly (though not exclusively) bound by SEP3 complexes lacking AP1 later in flower development. Indeed, among genes with the CArG box 2, we found an over-representation of GO categories involved in late reproductive processes, such as carpel, stamen, and anther development (see Additional file : Figure S4D). In summary, our data suggest that different CArG motifs are characterized by different footprint profiles and show temporal differences in their occupancy in flower development. The stage-specific enrichment of CArG motifs suggests a role in of combinatorial protein interactions in the spatiotemporal dynamics of AP1 and SEP3 DNA binding.

In agreement with previous findings [, ], we also identified GA-rich sequence motifs in the genomic regions bound by AP1 and SEP3 (see Additional file : Figure S4C). Candidate proteins that bind to this motif are among others the BASIC PENTACYSTEINE (BPC) transcriptional regulators, which control multiple aspects of plant development []. Recently it was shown that BPC proteins interact with MADS-domain proteins to regulate their target genes []. For this motif, footprints are most frequently detected in the day 2 and 4 datasets ( P ≤0.01, χ ² test), that is, during early stages of floral meristem development (Figure C). Thus, our data suggest a developmentally dynamic function of the GA-rich motif. However, its exact role and which factors bind to this motif remain to be determined.

MADS-domain TF DNA binding precedes changes in chromatin accessibility

In order to understand the dynamic relationship between chromatin accessibility and MADS-domain TF binding, we tested whether TF-bound genomic regions reside within DHSs (Figure ). At the earliest time point after floral induction, day 2, the vast majority of AP1- and SEP3-bound regions (73% and 68%, respectively) do not reside in DHSs. However, the overlap increases at later time points as development progresses, associated with a generally higher number of detected DHSs at later time points (Additional file : Table S5). At day 4, over 50% of the sites bound by AP1 or SEP3 reside in DHSs, a fraction that increases to about 75% at day 8 (Figure A). We see a significant relation between change in binding and accessibility of sites between 4 and 8 days. This relation is mainly explained by an over-representation of sites with both decreased binding and decreased accessibility (see Additional file : Table S7).

Under the hypothesis that MADS-domain TFs have a role in the modulation of chromatin accessibility, we should expect that quantitative changes in MADS-domain TF DNA binding should precede corresponding changes in chromatin accessibility during development (but not vice versa). In agreement with this idea, we found that increase in levels of DNA binding by AP1 or SEP3 from day 2 to day 4 correlates more strongly with corresponding changes in chromatin accessibility from day 4 to day 8, rather than simultaneous changes in accessibility from day 2 to day 4 (Figure B and see Additional file : Table S7). The same result was observed when we analyzed each biological ChIP-seq replicate independently (see Additional file : Figure S5). This delay in change in chromatin accessibility suggests that MADS-domain TFs may act as pioneer factors [] that directly or indirectly trigger changes in chromatin state during flower development. Among the genes for which AP1 and/or SEP3 may act as 'pioneer factors' are SUPPRESSOR OF OVEREXPRESSION OF CO 1 ( SOC1), SHATTERPROOF 2 ( SHP2), and GRF8 (Figure C). In all three gene loci at day 4, regions are bound by AP1 and/or SEP3, while these regions are hardly or not accessible but become accessible at a later time point. SOC1 is a special case since it is active in IMs, repressed in young floral meristems (stages 1 to 4) and later becomes expressed again in whorls 3 and 4, and it maintains expression during differentiation of stamens and carpels []. Also, the expression of SHP2 and GRF8 increases at later developmental stages (see Additional file : Table S4 and Figure D).

In conclusion, we observed that DNA-binding of AP1 and SEP3 can occur in chromatin regions that are not highly accessible, and that it can precede increase in DNA accessibility.

Plant development is controlled by the combined action of chromatin regulators and transcription factors. Here, we address the question of how this dynamic interplay is achieved at the molecular level using flower development as a model system. We characterize changes in MADS-domain TF occupancy, chromatin accessibility, and gene expression. Our results provide insights into the mechanisms by which MADS-domain TFs exert their master regulatory functions in meristem and organ differentiation in plants.

Developmental regulation of gene expression at the chromatin level

Data from the animal field show that developmental control of gene expression is linked with dynamic changes in chromatin accessibility. Given that multicellular development originated independently in plants and animals, we aimed to understand how dynamic the chromatin accessibility landscape is during plant development, and how this reflects changes in developmental gene expression. In summary, we observed changes in chromatin accessibility in the course of flower development, mostly in the transition from meristematic stages to floral organ differentiation. These changes can reflect the establishment of multiple new cell types during flower differentiation, and be linked with the activation of regulatory regions driving cell-type specific expression patterns of genes. It can also be related to the fact that during floral organ morphogenesis, gene activation is more frequent than downregulation of genes [, ]. Changes in DHSs globally correlate with changes in gene expression, although not all gene expression changes are associated with a change in chromatin accessibility. These findings suggest that there are multiple mechanisms by which developmental changes in gene expression are controlled, and that developmental changes in gene expression are partly manifested in changes in chromatin structure in plants.

MADS-domain TFs regulate target gene expression in a dynamic fashion

Although many MADS-domain TF-bound regions are occupied by these factors throughout flower development, we did observe dynamic quantitative changes in occupancy levels at a number of binding sites. Binding site dynamics reflect regulatory dynamics of genes with stage-specific functions in flower development, such as floral meristem patterning and organ growth. In line with previous results [, , ], our data suggest that floral MADS-domain TFs can act as repressors or as activators of gene expression. Given that many genes show no quantitative change in MADS-domain TF binding but they are differentially expressed throughout flower development, it appears that MADS-domain TF binding alone per se is not sufficient to explain changes in their gene expression, or that there is a delay in the regulatory response, for example, due to the mechanisms by which gene expression is regulated. It is possible that promoter binding by MADS-domain TFs is a prerequisite for regulatory response, but that additional factors are needed to generate cell-type or stage-specific gene expression patterns. This finding is supported by the fact that SEP3 and AP1, like other MADS-domain TFs, show relatively broad expression patterns in meristems and developing floral organs, and are thereby expressed in a variety of cell types, while the gene expression patterns of their targets need to be more tightly controlled, as we could show for GRF genes.

DNA-binding of MADS-domain TFs may trigger changes in chromatin accessibility

A result of the combined analysis of MADS-domain TF binding dynamics and chromatin accessibility is that MADS-domain TFs can select their binding sites independently of chromatin accessibility, and that binding of AP1 to DNA precedes local increase in chromatin accessibility. These results suggest that a mechanism by which AP1 regulates gene expression is through increasing accessibility of cis-regulatory regions. While this is the first report proposing such a mode of action for a plant TF, a similar mode of action has been previously described for animal TFs that trigger reprogramming of cell fate, such as Oct4, Sox2, Klf4, and c-Myc []. Previous results have shown that floral homeotic MADS-domain proteins form larger complexes together with ATP-dependent nucleosome remodelers and with histone-modifying enzymes in planta[, ]. Taken together, MADS-domain proteins may act as 'pioneer factors' that trigger changes in chromatin accessibility. Given the important roles of MADS-domain proteins as master regulators of developmental switches and floral organ specification, this is an intriguing mode of action. But how do these proteins target different regulatory regions at different stages of development? Based on the different properties of CArG boxes that we found for SEP3 and AP1, we propose that different higher-order MADS-domain protein complexes have different affinities for specific 'types' of CArG boxes. Thereby, changing MADS-domain TF occupancy at individual sites could modulate chromatin accessibility in a stage- or organ-specific manner.

In conclusion, our work represents a first step to a better understanding of the dynamics of regulatory networks in plants. By combining the information from DNA-binding and gene expression data, we are able to propose models of stage-specific regulatory interactions (Figure ). Our findings suggest that different homeotic factors regulate partly overlapping, yet also distinctive sets of target genes in a partly stage-specific fashion. Furthermore, MADS-domain TFs may regulate gene expression by alternative strategies, one of which is modulation of chromatin accessibility. Future research needs to reveal which target genes are specifically regulated by a certain homeotic protein complex, and by which exact molecular modes of action different sets of target genes can be modulated in specific ways.

Plant material

All plants were grown at 20°C under long day condition (16 h light, 8 h dark). Plants for ChIP-seq and DNase-seq were grown on rock-wool, whereas plants for gene expression analysis were grown on soil.

Tissue collection

For DNase-seq and ChIP-seq experiments: pAP1:AP1-GR ap1- 1 cal- 1 plants were dipped after bolting (2 cm to 5 cm height) in the DEX- induction solution (2 μM Dexamethasone, 0.01% (v/v) ethanol, and 0.01% Silwet L-77) daily. First induction was performed 8 h after lights on and daily induction at 4 h after lights on. Material was collected before DEX-induction, as well as at 2 days, 4 days, and 8 days after the first treatment (8 h after lights on). Two biological samples were generated for each time point. For gene expression profiling experiments: approximately 4-week-old pAP1:AP1-GR ap1-1 cal-1 plants were used. For each sample, inflorescence tissue from approximately 25 plants was collected using jeweler's forceps as previously described []. Four biologically independent sets of samples were generated for each experiment. For induction, inflorescences were treated with a DEX-induction solution, or with an identical mock solution that lacked dexamethasone. Using plastic pipettes, the solutions were directly applied onto the inflorescences so that the cauliflower-like structures were completely drenched. As for the DNase-seq and ChIP-seq experiments, after the first induction, daily induction was performed 4 h after lights on, and material was collected at the corresponding time-point 8 h after lights on. Material was collected immediately after solution application (0 days, mock), and at 2 days, 4 days, and 8 days after the first treatment.

DNase-seq experiments

Nuclei isolation was performed according to [] with minor modifications. Tissue was ground in liquid nitrogen. For each time point, 0.2 g of plant material was used. Ground material was resuspended in 2 mL of cold modified Honda buffer (HBM: 25 mM Tris, 0.44 M sucrose, 10 mM MgCl2, 10 mM β-mercaptoethanol, 2 mM spermine, and 0.1% Triton) and filtrated through a 55 μm membrane. The membrane was washed with 1 mL HBM buffer. The filtrate was applied to a sucrose 2.5 M/40% Percoll gradient and centrifuged 30 min 2,500 × g at 4°C. Nuclei were collected in the interphase and washed with 10 mL cold HBB (HBM without spermine) and 10 mL cold HBC (HBB with 20% glycerol). Between each wash, nuclei were centrifuged for 10 min 1,000 × g at 4C. DNA digestion was performed according to [] with minor modifications. Nuclei were resuspended in 2.5 mL buffer A (15 mM Tris-HCl (pH 8.0), 15 mM NaCl, 60 mM KCl, 1 mM EDTA (pH 8.0), 0.5 mM EGTA, 0.5 mM spermidine, and 11% sucrose) and divided into 12 1.5 mL tubes (aliquots of 200 μL). To each aliquot, 200 μL of 2× reaction buffer (Buffer A with 12 mM CaCl2, 150 mM NaCl) was added. Nuclei were mixed by inversion. DNase I was added (Roche Applied Science, Catalog #04716728001) to attain final concentrations of 110U-90U-70U-50U-35U-20U-15U-10U-7.5U-5U-2.5U-0U. Samples were incubated for 10 min at 37°C in a thermomixer. The DNase reaction was terminated by adding 400 μL of stop buffer (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.1% SDS, 100 mM EDTA (pH 8.0), 10 μg/mL Ribonuclease A, 1 mM spermidine, 0.3 mM spermine) and incubating at RT for 15 min. To each sample, 10 μL of 20 μg/mL proteinase K was added. After O/N incubation at 55°C, samples were centrifuged for 10 min at 13,000 × g. An aliquot of 10 μL of each sample were run on a 1% agarose gel. Samples that were not completely digested were selected for library preparation (Additional file : Figure S3E). DNA was precipitated by adding 0.9 volumes of isopropanol. The precipitated DNA was dried and left to resuspend in 100 μL HPLC water O/N at 4°C. DNA was purified with QIAGEN PCR purification kit (Cat. no. 28104). Two biological replicates for each time point were sequenced on Illumina HighSeq2000.

ChIP-seq experiments

ChIP experiments were performed following a previously published protocol [] using an anti-GR antibody (Glucocorticoid Receptor alpha Polyclonal antibody (PA1-516, Thermo Scientific), to precipitate AP1-GR), or a peptide SEP3 antibody []. 0.75 g of plant material were used for each biological replicate. ChIP experiments performed using pre-immuneserum were used as negative control for each time point. Two biological replicates for each experiment were sequenced on Illumina GAII or MiSeq.

DNase-seq and ChIP-seq data analysis

Base calling was performed using CASAVA version 1.7 for AP1 4 and 8 days ChIP-seq experiments days, while CASAVA version 1.8 was used for all the other analysis. Sequence reads reported by the Illumina's CASAVA v1.8 pipeline as low quality reads were removed from further analysis. CASAVA v1.7 does this automatically. FASTQ files were mapped to the Arabidopsis thaliana genome [] using Bowtie [] version 0.12.7, allowing up to three mismatches. Sequence reads mapped to mitochondrial and chloroplast chromosomes or mapping on multiple locations were removed. An overview of sequencing data is reported in Additional file : Table S8. Reproducibility between biological replicates was assessed using the Pearson correlation coefficient (PCC) for the genome-wide reads distribution at each pair of replicates on a single nucleotide resolution, for this, we used the script 'correlation.awk' provided by [], the results were: PCC >0.99 for DNase-seq experiments, and 0.80 < PCC < 0.977 for ChIP-seq experiments. Because of the high reproducibility of the data, FASTAQ files for replicates of the same experiment were combined. We used MACS 2.0.10 [] with default parameters except --mfold which was set to '2.20') to identify significant BSs for ChIP-seq experiments and significant DNase I hypersensitive sites (DHSs) for DNase-seq experiments. We used a cutoff of FDR ≤0.01 and FDR ≤0.001 (--qvalue parameter in MACS) for DNase-seq and ChIP-seq experiments, respectively. Genomic regions were associated with genes if located 3 kb upstream of the start of the gene up to 1 kb downstream of the end of the gene using the function distance2Genes in the Bioconductor package CSAR [] for genes annotated in TAIR10.

Quantitative comparison of ChIP-seq and DNase-seq experiments

We followed the Bardet et al. [] protocol for the peak alignment and normalization. Namely, we created an aggregated list of ChIP-seq and DHSs peaks in a region ± 75 bp around the peak summit, and then scored each one of those regions by the highest mapped read count normalized by total number of mapped reads in the library. This score was subsequently scaled by the score in the corresponding control sample in the same region. Quantile normalization implemented in the preprocessCore R package [] was then applied independently to all DNase-seq and to all ChIP-seq score values.

Changes in DHSs and putative TF BSs across the different time stages were quantified by means of (fold-change ratio). We classified regions as invariant when the fold-change was ≤ √2 for DNase-seq data and ≤2 for ChIP-seq data. Otherwise the region was classified as being an increasing or decreasing region according to the sign of the log2.

The simultaneous analysis of dependence between chromatin accessibility changes and TF binding changes, and of the influence of these factors on changes in gene expression (Figures E, C) was done by the chi-square test in Genstat 15 []. DNA sequences and overlapping regions were extracted using BEDTools [].

Motif analysis and DNase I cleavage

For motif identification, sequences of ChIP-seq peaks ± 50 bp around the peak summits, were submitted to MEME-ChIP [] after processed with RepeatMasker []; we used default parameters for MEME-ChIP except the motif site distribution ('-mod') parameter that was set to any number of repetitions ( anr). Motif occurrences were found in TF BSs (located 3 kb up to 1 kb downstream of genes annotated in TAIR10) using FIMO [] at P value <1e-5, and the DNase I cuts ± 100 bp around the motif matches at the same time stage were submitted to CENTIPEDE [] together with the proximity to the nearest TSS and the FIMO log-likelihood score ratio to infer TF binding by digital genomic footprinting. Then, each site was classified according to its posterior probability (pp) into three classes: footprint (pp ≥0.9), no footprint (pp ≤0.1), and unclear bound state (0.1 < pp < 0.9). For visualization of the average DNase I cleavage in Figure in a window ± 500 bp around the footprint, running-median smoothing was applied (width of median window equal to 5).

Information from The Plant Transcription Factor Database ([]) was used to identify overrepresented TF families. GO over-representation analysis was performed using the Cytoscape plugin BINGO [].

RNA preparation for microarray experiments

Total RNA was isolated from tissue samples using the Plant Total RNA kit (Sigma-Aldrich) according to the manufacturer's instructions. Quality of RNA samples was evaluated using a Bioanalyzer and a RNA Nano 6000 kit (Agilent). RNA concentrations were determined using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific).

Microarray set-up and experiments

Agilent microarrays were designed using the e Array software pipeline [] and TAIR genome annotation v8, and contain probes corresponding to 28,327 annotated genes (see []. Microarrays were used following manufacturer's instructions. RNA samples were labeled with fluorescent dyes using the Quick Amp Labeling Kit (Agilent). Microarray hybridizations (65 C, 16 h) and washes were performed with Agilent reagents and following standard protocols. Microarrays were scanned using an Agilent DNA Microarray Scanner, and data were acquired using Agilent's Feature Extraction Software. Four independent sets of biological samples were used for the experiments. The dyes used for labeling RNA from a given time point were switched in the replicate experiments to reduce dye-related artifacts. Samples were co-hybridized as follows: 0 days to 2 days, 2 days to 4 days, and 4 days to 8 days, resulting in a total of three hybridizations per set, and two biological replicate sets labeled with each dye polarity.

Gene expression microarray data analysis

Feature extraction software pre-processed data from the Agilent microarrays were imported into the Resolver gene expression data analysis system version 7.1 (Rosetta Biosoftware, Seattle, WA) and processed as described []. Resolver uses a platform-specific error model-based approach to stabilize the variance estimation to improve the specificity and sensitivity in differential gene expression detection []. The data from the four biological replicates of each condition were combined, resulting in an error-model weighted average of the four. The P values for differential expression calculated by Resolver were adjusted for multi-hypothesis testing using the Benjamini & Hochberg procedure, as implemented in the Bioconductor multtest package in R []. Genes for which the Benjamini & Hochberg-adjusted P value was <0.05 in at least one of the comparisons (that is, time points), and that passed an absolute fold-change (FC) cutoff of 1.8, were considered as differentially expressed (see Additional file : Table S4). Genes that were detected as differentially expressed were subjected to cluster analysis using the k-means algorithm implemented in Resolver (partitioning into different numbers of clusters was tested, and k = 6 was selected for producing the most consistent clusters (Figure B).

Isolation of RNA and real-time PCR analysis

Total RNA was extracted using Invitek Kit according to the manufacturer's protocol. DNase I digestion was performed on total RNA using DNase I from Invitrogen. RNA integrity was checked on 1% (w/v) agarose gels before and after DNase I treatment. Absence of genomic DNA was confirmed subsequently by qRT-PCR using primers, which amplify an intron sequence of the gene At5g65080 (Forward 5′-TTTTTTGCCCCCTTCGAATC-3′ and reverse 5′-ATCTTCCGCCACCACATTGTAC-3′). First-strand cDNA was synthesized from 4 μg of total RNA using TaqMan kit (Roche) cDNA Synthesis Kit following the manufacturer's protocol. The efficiency of cDNA was estimated by qRT-PCR using two different primer sets annealing 5′- and 3′- ends of a control gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (At3g26650), respectively, (GAPDH3′: forward 5′-TTG GTG ACA ACA GGT CAA GCA - 3′ and reverse 5′-AAA CTT GTC GCT CAA TGC AAT C-3′) (GAPDH5′: forward 5′-TCT CGA TCT CAA TTT CGC AAA A - 3′ and reverse 5′-CGA AAC CGT TGA TTC CGA TTC -3′). Transcript levels of each gene were normalized to ACTIN2 gene (5′- TCCCTCAGCACATTCCAGCAGAT-3′ and reverse 5′-AACGATTCCTGGACCTGCCTCATC-3′). Large-scale qRT-PCR for 1,880 TFs was performed as described previously [, ], using an ABI PRISM 7900HT sequence detection system (Applied Biosystems Applera, Darmstadt, Germany). Amplification products were visualized using SYBR Green (Applied Biosystems).

MIR396 constructs and GFP fusion reporter gene constructs

35S:miR396a was generated by fusing 400 bp of MIR396a precursor to the 35S promoter in the pCHF3 binary plasmid []. ANT: miR396a was generated by replacing the 35S viral promoter in the previous vector with the ANT promoter (5.8 kb upstream regulatory sequences) [].

AtGRF2, AtGRF5, AtGRF7, and AtGRF8 genomic regions were amplified by PCR using the following primers: AtGRF2, fw: 5′-AACATTTGGTTGGTAATGTCAGCGT-3′ rev: 5′-GGTTGTGTAATGAAAGTAATCGCCA-3′, AtGRF5, fw: 5′-GTATGTTCAAATAATGTGAATCGTGG-3′ rev: 5′-GCTACCTGAGAAAATAAATTTAAACT-3′ AtGRF7, fw: 5′-GAATCTTGTTCTTCAGAAAGATGAAC-3′ rev: 5′-AACCTGGCTGCTTTCGTCGGAC-3′ and, AtGRF8, fw: 5′-GTTTGTTTGTTACATTGCCGTTT-3′ rev: 5′-GCTTGAGCTTCTGCTGCA-3′. The PCR fragments were cloned into the GATEWAY vector pCR8/GW/TOPO from Invitrogen and transferred via LR reaction into the destination vector pMDC107 []. Expression vectors were introduced into Arabidopsis thaliana ecotype Col-0 by floral dip transformation []. Transformant plants were select on MS medium with Hygromycin (10 ug/mL).

Confocal Scanning Laser Microscopy (CSLM)

GFP tagged protein localization was observed trough CSLM on Leica SPE DM5500 upright microscope using a ACS APO 40x/1.15 oil lens and using the LAS AF 1.8.2 software. FM4-64 dye was added to 0.1% agar at a concentration of 5 M and used as staining for cell membranes. GFP and FM4-64 dye were excited with the 488-nm line of an Argon ion laser. The GFP emission was detected at a bandwidth of 505-530 nm, while FM4-64 dye and chloroplast auto fluorescence were detected at a bandwidth of 650 nm. After acquisition optical slices were median filtered and three-dimensional projections were generated with LAS AF 1.8.2 software package.

Accession numbers

Microarray data have been deposited with the NCBI Gene Expression Omnibus (GEO) under accession number GSE47981. ChIP-seq and DNase-seq data have been deposited under accession number GSE46986 and GSE46894, respectively.

Acknowledgements

The authors would like to thank Christa Lanz, Markus Schmid, and Elio Schijlen for generating the Illumina GAII and Hiseq data, and Marjolein van Eenennaam and Marco Busscher for generating of the GRF8-GFP construct. We are grateful to Oriol Casagran for help with microarray hybridizations. Some parts of the computations were made at the Poznań Supercomputing and Networking Center. This work was supported by an NWO-VIDI grant to KK, and a Marie-Curie-ITN network grant SYSFLO (FP7/2007-2011, grant agreement no. 237909) to AP, PM, PK, and GCA. KK wishes to thank the Alexander-von-Humboldt foundation for support. This work was also supported by grants from Spanish Ministerio de Ciencia e Innovación (BFU2011-22734 to JLR). JTM and JJ were recipients of EMBO postdoctoral fellowships.

Electronic supplementary material

Competing interests

Authors' contributions

AP performed the ChIP experiments, DNaseI-seq experiments, generated and analyzed the marker lines, and drafted the manuscript. PM and JMM were responsible for bioinformatic analysis; AP and PM interpreted bioinformatics results; JTM and JJ performed the microarray studies and the data analysis; SB and MA helped with the transcriptome analysis; MAM, JMD, JP, and DSOM performed biological analysis; AP, FW, PK, and JLR participated in the design of the study and in the data analysis, and drafted the manuscript. GCA and KK conceived of the study, supervised and coordinated the study and drafted the manuscript. All authors carefully read and approved the final manuscript.

The authors declare that they have no competing interests.

Below are the links to the authors' original submitted files for images.

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Dynamics of chromatin accessibility and gene regulation by MADS-domain transcription factors in flower development

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Development of eukaryotic organisms is controlled by transcription factors that trigger specific and global changes in gene expression programs. In plants, MADS-domain transcription factors act as master regulators of developmental switches and organ specification. However, the mechanisms by which these factors dynamically regulate the expression of their target genes at different developmental stages are still poorly understood.

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References

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Shaping the Arabidopsis transcriptome through alternative splicing. Adv. Bot. 2015, 419428 (2015).

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Acknowledgements

Research reported in this publication was supported by the Agence Nationale de la Recherche (Projet FLOPINET), Centre National de la Recherche Scientifique funding to S.J.C., NHMRC project grant funding (GNT1089167) to S.J.C., Australian Research Council Future Fellowship (FT160100318) to S.J.C., ATIP-Avenir and LabEx GRAL (ANR-10-LABX-49-01) program funding to C.Z., Premier's Research and Industry Fund grant provided by the South Australian Government Department of State Development to V.T. and CEA Irtelis fellowship to A.N. Mouse monoclonal S9.6 antibody was kindly donated by S. Leppla (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA).

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Affiliations

1. Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen 72076, Germany

2. Laboratory of NF-κB Signaling, Institute of Molecular and Cell Biology (IMCB), 61 Biopolis Drive, Proteos, Singapore 138673, Singapore

3. Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore (NUS), Singapore 117597, Singapore

4. Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, SE-901 87 Umeå, Sweden

Contributions

S.J.C. and C.Z. conceived the project. V.M.C., S.J.C., V.H., A.N., S.A.C., Gi.C., Go.C., A.J., V.T. and M.S. performed all experiments, provided material and analysed the results. S.J.C. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Simon J. Conn.

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A circRNA from SEPALLATA3 regulates splicing of its cognate mRNA through R-loop formation

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Transgenic Banana Plants with Reduced Expression of MaMADS1 or MaMADS2

Banana fruit start to produce ethylene in the pulp and later in the peel ( Supplemental Fig. S1), indicating that ripening is initiated in the pulp. We previously described the expression of six MaMADS box genes from banana fruit ( Elitzur et al., 2010), and based on RNA sequencing (RNAseq) data, we discovered seven additional highly expressed MaMADS box genes (more than 50 reads per kilobase of transcript per million reads mapped [ RPKM]) in the pulp of banana. Homologs of SlFUL1/2, SlTAGL1, and SlMADS-RIN were identified and used to construct a phylogenetic tree ( Supplemental Fig. S2). MaMADS2 and MaMADS1 are most similar to SlMADS-RIN, with 62% and 56% amino acid identity, respectively ( Elitzur et al., 2010).

We proceeded to functionally characterize the MaMADS1 and MaMADS2 genes, whose expression is highly elevated in the pulp in concert with ripening. Furthermore, MaMADS2 expression was not affected by ethylene in the pulp, suggesting that at least MaMADS2 expression may precede (and thus mediate) the induction of key ripening processes. These MaMADS box genes contain four domains: M, I, K, and C, with the M domain most conserved. The C region is the most divergent, as is typical of MADS box genes ( Elitzur et al., 2010); therefore, all three repression constructs targeted the C region ( Supplemental Fig. S3). Specifically, three repression constructs were created: (1) the 195-bp RNAi MaMADS1 targets the K and C domains; (2) RNAi MaMADS2 targets the C domain and the 3′ untranslated region ( UTR; 302 bp); and (3) antisense ( AS) MaMADS2 targets the same sequence as for (2). The construct used for MaMADS1 silencing has 61% homology with MaMADS2, and the longest stretch of identity is 11 nucleotides. The construct used for MaMADS2 silencing has only 38% homology with MaMADS1, and the longest stretch of identity is seven nucleotides. Identities of up to 51% with stretches of up to 12 nucleotides resulted in gene-specific silencing in a gene family in rice ( Oryza sativa; Miki et al., 2005). The above DNA targets are depicted in Supplemental Figure S3 and detailed in "Materials and Methods." The constructs were used for the transformation of banana embryo cultures, and transgenic plants were recovered as described in Table I. Twenty-six plants were recovered from RNAi MaMADS1 transformation, 19 from RNAi MaMADS2, and 18 from two independent transformations of AS MaMADS2. The transformation of all three constructs yielded fertile plants verified for inserts using leaf DNA as described in "Materials and Methods" with no obvious growth phenotype.

Banana fruit were harvested from independent transgene-confirmed plants of all three transgenic classes ( RNAi MaMADS1, RNAi MaMADS2, and AS MaMADS2), in addition to wild-type untransformed control plants propagated at the same time. Target gene mRNA levels at the breaker (color transition) stage in peel and pulp were determined by quantitative real-time ( Q-RT)-PCR ( Fig. 1). MaMADS1 or MaMADS2 mRNA accumulation in control plants was high in peel and pulp, as expected from prior results ( Elitzur et al., 2010). MaMADS1 mRNA was low in peel and pulp of RNAi MaMADS1 lines, and MaMADS2 mRNA was low in both RNAi MaMADS2 and AS MaMADS2 peel and pulp for all transgenic plants examined. The specificity of the respective silencing constructs was examined by determining MaMADS1 mRNA expression in MaMADS2-suppressed fruit and MaMADS2 mRNA in MaMADS1-suppressed fruit. Down-regulation of MaMADS1 did not substantially reduce the levels of MaMADS2 in the pulp but did reduce it in the peel, suggesting a regulatory rather than transgene homology effect in the peel. Down-regulation of MaMADS2 substantially induced MaMADS1 in the pulp at the breaker stage but reduced it in the peel in both RNAi MaMADS2 and AS MaMADS2 fruit. The reduction of the target genes in both peel and pulp, yet tissue-specific repression of the alternate gene (and induction of MaMADS1 in MaMADS2 repression lines), are consistent with a regulatory repression rather than a transgene homology effect.

MaMADS1 and MaMADS2 Are Both Necessary for Ripening

In order to ascertain the impact of MaMADS1 or MaMADS2 repression on ripening, we monitored transgenic and control fruit prior to and through ripening during postharvest storage at room temperature. Wild-type banana fruit ripened 12 to 13 d after harvest, while MaMADS1- and MaMADS2-suppressed fruits were delayed in the ripening transition by 3 to 14 d ( Supplemental Table S1). Transgenic and control fruit (fingers) and hands (banana finger clusters) are shown in Figure 2, where at 20 d postharvest the control fruit show severe decay, while transgenic fruit remain yellow or green with little damage even at 26 d ( Fig. 2). Fruit color, firmness, and total soluble solids ( TSS) also were measured in the fruit of wild-type and transgenic plants ( Fig. 3; Supplemental Table S1). In general, in all fruits of transgenic plants, breaker occurred later than in controls. More specifically, changes in color in the control plants occurred following the ethylene production burst ( Figs. 3 and 4), while in transgenic plants, especially those that did not produce ethylene, it occurred either following or concomitant with the increase in respiration. At this stage, fruits of transgenic plants had higher firmness than wild-type fruit. These results demonstrate a strong influence on both time to onset of ripening and shelf-life of the MaMADS1 and MaMADS2 transcription factors. TSS of the peel and pulp were, in most lines, similar to those in the wild type yet correlated with the delayed-ripening transgene effects. These results further demonstrate the utility of using these genes for extending shelf-life across all important quality phenotypes, similar to the effects of the widely used rin mutation for tomato shelf-life extension.

MaMADS1 and MaMADS2 Affect Ethylene and Respiration

To further define the effects of these banana MADS box genes on ripening, respiration and ethylene production were measured on control and transgenic fruits at consecutive days from harvest until the appearance of brown spots ( Fig. 4). In each of the control I groups, typical climacteric respiration occurred at 9 to 12 days after harvest ( DAH), preceding the expected burst in ethylene production (12-15 DAH; Fig. 4A). Fruit of two independently transformed RNAi MaMADS1 plants were analyzed, and in both, respiration and ethylene were delayed or attenuated ( Fig. 4B). These observations confirm the necessity of MaMADS1 for ripening via the control of climacteric respiration and ethylene hormone synthesis.

The development of plant chimeras with the transgene further corroborated the involvement of MaMADS1 in fruit ripening. Besides the banana bunches harvested from the lines mentioned above, a number of bunches from other lines were not uniform in their color development after harvest, and bananas derived from the fifth hand exhibited earlier ripening than bananas from upper (older) hands ( Fig. 5A). To further elucidate this phenomenon, the DNA of banana from different hands of the same bunch of three different lines was examined ( Fig. 5B). It can be seen that, in three plants that harbor the correct insert (as judged by comparison with leaf PCR), no inserts was observed in the fifth hand, and only in two lines was an insert observed in the second hand (the control gene was amplified in all samples; data not shown), suggesting that the transformation yielded some chimeric plants. These results were in agreement with ethylene and carbon dioxide production for fruits of different hands ( Fig. 5C). In control fruits, the ethylene production peak occurred on day 12 after harvest, even later than in bananas that did not harbor the insert (11 d in the second hand of line 21 and 7 or 9 d in the fifth hand of lines 21 and 18 or 8, respectively). However, the ethylene and carbon dioxide peaks appeared on day 16 after harvest in fruits that harbor the insert (second hand of lines 8 and 18).

Fruits from RNAi MaMADS2 transgenic plants also showed reduced respiration and ethylene production ( Fig. 4, C and D), while even greater inhibition of ethylene production and respiration was observed in banana fruit from AS MaMADS2 transgenic plants ( Fig. 4E). In two AS MaMADS2 plants (AS 40 and AS 45), the fruit did not show any increase in ethylene, and in these fruits, respiration was attenuated and increased gradually starting 26 DAH. In two additional plants, the ethylene production peak was lower than that of controls and occurred later than in the control (10 DAH, control; 18 DAH, AS 36; and 15 DAH, AS 37). These ethylene peaks occurred in parallel with increased respiration.

Repression of MaMADS1 or MaMADS2 Modified the Expression of Other MaMADS Box Genes

To further assess the effects of transgene suppression, we determined the levels of expression in MaMADS1- and MaMADS2-repressed lines of MaMADS3 to MaMADS5 ( Fig. 6). MaMADS3, a SEP gene with lower identity to MaMADS1/2 (52% and 54% identity to MaMADS1 and MaMADS2, respectively), showed reduced expression in both types of transgenic plants in comparison with wild-type fruit at breaker in peel. The possibility of MaMADS3 targeting, by either transgene or both transgenes, remains a formal possibility, although the lower level of homology of this gene to MaMADS1 or MaMADS2 sequences used for silencing (39% and 28.5%, respectively) would argue against direct transgene effects and suggests that MaMADS3 is under regulatory constraints imposed by both genes. On the other hand, the levels of both MaMADS4 (a SEP gene) and MaMADS5 (an AG gene) increased in the pulp of MaMADS2-repressed plants. The levels of MaMADS5 also increased in the pulp of MaMADS1-repressed plants ( Fig. 6). These results suggest that MaMADS4 and MaMADS5 genes are under negative control of MaMADS2 and MaMADS5, possibly also under the negative control of MaMADS1 in the pulp. Alternatively, these results represent a compensatory mechanism. They are consistent with regulatory rather than direct transgene effects. The up- and down-regulation of additional MADS box genes is consistent with reports in tomato, where a number of MADS box genes are repressed in the rin mutant ( Martel et al., 2011; Zhong et al., 2013) and some are repressed and others are induced in transgenic TAGL1 repression lines ( Itkin et al., 2009; Vrebalov et al., 2009). These altered expression patterns in both banana and tomato represent downstream regulatory effects or compensatory mechanisms. While the specific functions of some of these responses to reduced-ripening MADS box gene expression remain to be determined, in no instance do they detract from the mutant- or transgene-demonstrated necessity of genes such as MaMADS1 or MaMADS2 for normal ripening manifestation.

MaMADS1- and MaMADS2-Repressed Banana Fruit Retain Ethylene Sensitivity

It was clear that normal ripening was delayed in fruit of the transgenic plants, and it was important to determine how these fruit respond to ethylene treatment, both to clarify their ripening responses and to enable their practical utilization, as ripening-delayed fruit require ethylene responsiveness. Banana fruit of the control and transgenic plants were exposed to ethylene. Fruit of the MaMADS-repressed lines responded to ethylene in a similar manner to control fruit and developed yellow color ( Fig. 7A). In addition, treatment with ethylene increased respiration and ethylene production ( Fig. 7B), at least in one AS MaMADS2 line (line 37). Further examination of both the firmness and color in fruits from several repressed lines showed that there were no differences between the wild type and any of the transgenic plants following ethylene treatment ( Supplemental Fig. S4). These results demonstrate that, while MaMADS1 and MaMADS2 are necessary for endogenous ethylene production and ripening, ripening-delayed fruit repressed in either gene are still responsive to ethylene treatment necessary for optimal market utility and performance.

Ethylene Alters the Expression of Multiple MaMADS Box Genes in MaMADS2-Repressed Fruit

To further clarify if the MaMADS2-repressed lines respond to ethylene by modifying fruit MaMADS box gene expression patterns, we analyzed multiple fruit MaMADS box genes that are homologs to genes known to affect ripening in tomato. Banana fruits of AS MaMADS2 (line 37) and the control were treated at harvest with ethylene, tissue was collected immediately following ethylene treatment, and the levels of fruit MaMADS box genes' expression were compared between ethylene-treated and nontreated wild-type and AS MaMADS2 (line 37) plants. The expression of MaMADS1, MaMADS4, and MaMADS9 was induced by ethylene in both the control and the repressed line, and MaMADS5 was repressed by ethylene in the MaMADS2 line, although to levels still approximately 50% higher than in controls with or without ethylene. MaMADS7, MaMADS8, MaMADS10, and MaMADS11 showed little change in response to either MaMADS2 reduction or ethylene. Whether ethylene confers ripening in MaMADS2-repressed lines in the context of residual MaMADS2 or via other functionally redundant MaMADS gene(s) remains to be determined, as does the relevance of the altered expression of additional MaMADS box genes due to reduced MaMADS2 expression or ethylene. The induction of MaMADS1 by ethylene remains consistent with the possibility that MaMADS1 and MaMADS2 are functionally equivalent, although the clear ripening-delay phenotypes resulting from either MaMADS1 or MaMADS2 repression confirm that wild-type levels of both genes are necessary for ripening, absent any external influence such as the application of exogenous ethylene. Testing the potential functional redundancy would require specific induction of MaMADS1 (presumably via a transgene) in MaMADS2-repressed lines.

MaMADS1 and MaMADS2 Are Necessary for Banana Ripening

Much of our knowledge of fleshy fruit ripening is derived from the model dicot tomato, and it is not clear whether all fruit, including those of monocots like banana, employ similar genetic mechanisms. Tomato, however, does provide numerous candidate genes for the identification and testing of functional homologs in other important fruit species. One such well-studied tomato gene is SlMADS-RIN, whose mutation is widely used for tomato ripening control and shelf-life extension ( Vrebalov et al., 2002), validating the practical value of regulating this ripening factor in a commercially viable manner. Here, we show, through transgene-mediated silencing, that the MaMADS box genes MaMADS1 and MaMADS2 are necessary genetic components of ripening. Furthermore, as ethylene is induced in pulp prior to peel, we suggest that changes occurring in pulp are central to initiating fruit ripening and result in a cascade of hormone and additional transcription factor activities that mediate the full process in both peel and pulp. As an example, MaMADS3, which shows very low homology to either MaMADS1 or MaMADS2, is repressed in peel of both MaMADS1- and MaMADS2-repressed lines due to the reduction in ethylene occurring in these fruit. Its expression is recovered with ethylene, as shown previously ( Elitzur et al., 2010).

The specificity of transgene silencing is supported by the facts that the expression of other MaMADs box genes either did not change or was altered in a tissue-specific manner, inconsistent with targeted transgene repression ( Figs. 1 and 6) and more likely reflecting the regulatory interactions among ripening MADS box genes also reported in tomato ( Zhong et al., 2013). How these additional regulators participate in the ripening cascade requires further investigation. In contrast to the single tomato SEP regulator SlMADS-RIN, in banana, both E class ( SEP) MaMADS1 and MaMADS2 genes are necessary for ripening. The central ripening function of the SEP MADS box genes is clearly an ancient activity of this gene family, as it is common to both monocots and dicots, although evolution has tailored common regulators to distinct fruit outcomes.

Reducing the transcript levels of either MaMADS1 or MaMADS2 ( Fig. 1) decreased ripening progression, as was demonstrated by the inhibition in color change, delayed softening, and slower accumulation of sugar in the pulp and peel ( Figs. 2 and 3). The fruits of transgenic banana lines, once ripened, had similar characteristics to control fruits, which matured many days earlier ( Fig. 3; Supplemental Fig. S1). This phenomenon likely reflects the activity of residual target gene activity in all transgenic repression lines and is supported by a similar dose response in tomato lines heterozygous for the rin mutation. Silencing either gene delayed and reduced ethylene production or completely abolished it. However, climacteric respiration (as determined by carbon dioxide evolution), although delayed in MaMADS1- and MaMADS2-suppressed fruits, eventually reached wild-type fruit rates, even in lines that never demonstrated elevated ethylene synthesis ( Figs. 3 and 4). The phenotypes of the described transgenic banana plants indicate that both the MaMADS1 and MaMADS2 genes are high-value targets for banana ripening and shelf-life improvement.

MaMADS2 Acts Upstream in the Ripening Cascade

MaMADS2 expression increases at the onset of ripening yet was not modified in peel and pulp by ethylene, indicating that it may act upstream of this hormone's ripening-related synthesis ( Elitzur et al., 2010). The fact that ethylene synthesis and ripening is delayed upon MaMADS2 repression is consistent with this hypothesis. Moreover, MaMADS2 negatively regulates the expression of MaMADS4 and MaMADS5 in pulp at breaker and at harvest ( Figs. 6 and 7C) and, to a lesser degree, MaMADS10 and MaMADS11 (homologs of FUL1 and FUL2) at harvest ( Fig. 7C) and MaMADS1 at breaker ( Fig. 6), at the time of its induction ( Elitzur et al., 2010). MaMADS2 expression in pulp was not affected in MaMADS1-repressed fruit ( Fig. 1), confirming a regulatory hierarchy where MaMADS2 has influence over MaMADS1, but the reverse seems not to be true, at least in the context of the available data. It is possible that residual MaMADS1 and MaMADS2 expression observed in even the most strongly repressed lines contributes to the fact the transgenic bananas eventually reached full ripening, unlike tomatoes harboring the loss-of-function SlMADS-RIN gene mutation ( rin) but similar to heterozygous rin/RIN lines used widely in commercial production ( Vrebalov et al., 2002). It remains a possibility that the increase in expression of other MaMADS box genes observed in MaMADS2-repressed lines, especially MaMADS1 but possibly others ( MaMADS4, MaMADS5, MaMADS10, and/or MaMADS11; Figs. 1, 6, and 7C), may contribute to the partial ripening observed in these lines. The possible contributions of these genes remain to be determined, but the data here further support an upstream role of MaMADS2 in normal banana fruit maturation.

Ethylene May Induce Ripening in MaMADS2-Suppressed Lines by Affecting Additional Banana MADS Box Genes

The induction by ethylene of several SEP MADS box genes ( MaMADS1 and MaMADS4) and AG ( MaMADS9) in the AS MaMADS2-suppressed line ( Fig. 7C) may contribute to the recovery of ripening in response to exogenous ethylene. The substantial elevation in MaMADS5 mRNA in response to MaMADS2 repression is interesting, as is its repression by ethylene at harvest in these same lines ( Fig. 7C). MaMADS5 is most similar to banana MaMADS7 (88% identity), which accelerates ripening when expressed in tomato ( Liu et al., 2015a). MaMADS5 and MaMADS7 are most similar to tomato SlTAGL11, which is expressed in fruit ( Busi et al., 2003) but for which no phenotype of suppressed lines has been reported. The closest homolog in banana of the tomato ripening gene SlTAGL1 ( Itkin et al., 2009; Vrebalov et al., 2009) is MaMADS9 (67% identity). In tomato, SlTAGL1 expression is not influenced by the loss of SlMADS-RIN activity in the rin mutant ( Vrebalov et al., 2009), nor is MaMADS9 altered when MaMADS2 is repressed ( Fig. 7C). Nevertheless, while MaMADS9 expression is induced by ethylene in the wild type and in the mutant, the SlTAGL1 transcript is not affected by ethylene ( Itkin et al., 2009; Vrebalov et al., 2009), indicating a divergence of precise regulatory signals for these genes in the two species.

Our results suggest the conservation of multiple ripening MADS box gene functions between fleshy fruits of the monocot banana and the dicot tomato. Nevertheless, the functions of specific orthologs likely diverged in the context of differing fruit physiologies and morphologies presented by banana and tomato.

MaMADS1 and MaMADS2 Are Logical Targets for Banana Ripening Control

Taken together, the repression of either MaMADS1 or MaMADS2 results in delayed ethylene synthesis and ripening, although the fruit eventually achieve normal ripening based on an assessment of typical industry and market traits. Moreover, suppressed lines remain sensitive to ethylene, making them suitable to the market practice of ethylene treatment to mature green bananas. It is noteworthy that tomato hybrids heterozygous for the SlMADS-RIN mutation ( rin) are prevalent in the marketplace, conveying inhibited ethylene responsiveness and long shelf-life in the absence of costly postharvest inputs. MaMADS1 and MaMADS2 repression phenotypes in banana highlight the potential for similar application to an enormous food crop important for developing country food security and international commerce. The functional utility of these genes in such diverse taxa suggests that similar genes will likely be effective targets for fruit storage and shelf-life improvement in numerous crop species.

Plant Material

Banana ( Musa acuminate, AAA Cavendish subgroup, Grand Nain) was used for transformation, and the resulting plants were planted in Western Galilee, Israel. Fruit characteristics were determined in parallel for transgenic and nontransformed control plants grown in the same field. Since fruits of transformed plants were harvested at different times, control fruits were harvested together with the transgenic fruits. Fruit were harvested at the commercial stage (75% final cross-sectional filling), and their maturity and developmental stage were evaluated by determining the average of the locular angles of banana fruit and the ratio of peel to pulp in cross sections ( Dadzie and Orchard, 1997). Hands of the first, second, or third tiers containing 10 to 30 fruit were separated from the bunch to monitor the harvest, preclimacteric (green), climacteric (color break), and postclimacteric (appearance of brown spot) stages. Following separation, the cut area of the hands was sprayed with 0.1% (w/v) thiobendazole to prevent crown rot decay, and the hands were air dried, packed in perforated polyethylene bags and stored at 20°C and 95% relative humidity. Pulp and peel samples of at least four fruits were taken separately and frozen on consecutive days, up to 35 DAH. A subset of fruits was treated with ethylene for 20 h from 1 to 4 DAH to assess the ethylene response. Ethylene at 1 µL L −1 elicited a response; however, in some cases, fruits were treated with 10 µL L −1, and details are given in the figure legends.

Measurements of Ripening Parameters

Ethylene and carbon dioxide production was measured during postharvest at 20°C. These gases were determined by gas chromatography head space gas analysis of individual banana fingers in 2-L sealed glass jars at 20°C as described ( Elitzur et al., 2010). Peel color was determined from the surface area of three individual banana fingers using a Minolta CR-300 colorimeter. Firmness was measured in the middle of whole fruits using a Chatillon Force tester (Ametek). TSS of peel and pulp juice, resulting from freezing and thawing of the tissues, was determined using a hand-held HSR-500 refractometer (Atago).

Construction of Plasmids for MaMADS1 and MaMADS2 Repression

Gene sections used for RNAi or AS constructs for MaMADS1 (EU869307/Ach11P19540) or MaMADS2 (EU869306/Ach9P01370) are described in Supplemental Figure S3. Table I summarizes the transgenes created. The plasmids and the correct inserted DNA are described in Supplemental Figure S5. All insertions were under the control of the constitutive 35S promoter. Specifically, an AS construct of MaMADS2 was created by cloning the 303-bp C domain and 3′ UTR spanning nucleotides 520 to 822 of the MaMADS2 complementary DNA ( cDNA; GenBank accession no. EU869306) in reverse orientation into a modified pBIN binary vector, pBIN117 (kindly provided by Rahan Meristem). The plasmid was generated by XhoI and EcoRI linker insertion at the NotI restriction site of the pBIN117 multiple cloning site. Cloning was facilitated by target sequence PCR using forward and reverse primers containing the XhoI and EcoRI restriction enzymes sites, respectively (for primer sequences, see Supplemental Table S2). The resulting plasmid contains the NPTII gene under NOS promoter control for kanamycin selection.

Gateway technology (Invitrogen) was used for the RNAi constructs. MaMADS1 from base 333 to 528 (GenBank accession no. EU869307), representing the partial K and C domains, was cloned by site-specific recombination into pHellsgate2 (GenBank accession no. AJ311874), and MaMADS2 from base 520 to 822 (GenBank accession no. EU869306), spanning the C domain and 3′ UTR, was cloned into pHellsgate8 (GenBank accession no. AF489904), kindly provided by Commonwealth Scientific and Industrial Research Organization Plant Industry. The MaMADS1 and MaMADS2 repression sequences were amplified from banana fruit cDNA using the primers described in Supplemental Table S2, which included the attB1/attB2 recombination sites. The resulting PCR products were gel purified using the QIAquick PCR purification kit (Qiagen) and cloned into pDONR221 (Invitrogen; catalog no. 12536-017), using Gateway BP Clonase II Enzyme Mix (Invitrogen; catalog no. 11789-020) mediated by the attB sites of pDONR. A second Clonase step used LR Clonase II Enzyme Mix (Invitrogen; catalog no. 11791-020) mediated by the attL sites created on pDONR and the attP or attR sites of the entry vector, which exist in pHellsgate2 and pHellsgate8, respectively ( Supplemental Fig. S5). The plasmids were verified by restriction enzyme digestion, PCR, and sequencing of PCR products from each plasmid.

Following transformation, the presence of the constructs was verified by PCR on leaf DNA using the Extract-N-Amp Plant PCR Kit (Sigma-Aldrich). The reactions and the expected products are described in Supplemental Figure S5, with primers listed in Supplemental Table S3 and resulting PCR product images shown in Supplemental Figure S5C. Negative control fruit contained no plasmid sequences.

Transformation of Embryonic Banana Cultures

Immature male flowers, approximately 3 d postemergence, were used for the generation of embryonic callus. Male flowers were harvested and treated as described ( Strosse et al., 2003). Resulting calli were transferred to an embryonic cell suspension ( Schoofs, 1997). Cell clumps were used for transformation by Agrobacterium tumefaciens with the inclusion of kanamycin. Transformed somatic embryos were transferred to a semisolid medium containing one-half-strength Murashige and Skoog medium and 10 µm zeatine for approximately 6 months until shoots were clearly visible. Plantlets were hardened under mist in a greenhouse for approximately 6 weeks, until the fourth leaflet emerged, and then transferred to the field ( Table I).

Determination of Gene Expression by Q-RT-PCR Analysis

Total RNA was extracted using the Spectrum Plant Total RNA Kit (Sigma-Aldrich) and treated with TURBO DNase free (Applied Biosystems) as described by the manufacturer. First-strand DNA was prepared using the Verso cDNA Kit (Thermo Fisher Scientific) and used for Q-RT-PCR analysis. Primers ( Supplemental Table S4) were designed with Primer Express version 2 (Applied Biosystems). Primer concentration was 4 to 8 µm, and the cDNA was diluted for PCR of the gene of interest and reference RIBOSOMAL RNA ( rRNA) and GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE ( GAPDH) genes (GenBank accession nos. EU433925 and AY821550, respectively). Template concentrations were determined as described ( http://www.abgene.com/downloads/article-SYBRoptimise.pdf.). Forward and reverse primers for the references genes are 5′-GCAAGGATGCCCCAATGT-3′ and 5′-AGCAAGACAGTTGGTTGTGCAG-3′ for GAPDH and 5′-GCGACGCATCATTCAAATTTC-3′ and 5′-TCCGGAATCGAACCCTAATTC-3′ for rRNA. Reaction mixtures contained cDNA, forward and reverse primers, and Power SYBR Green PCR Master Mix (Applied Biosystems) in a 20-µL total volume. Reactions were run in triplicate on a Rotor-Gene 3000 PCR machine (Corbett Life Research) using 35 cycles of 95°C for 10 s, 60°C for 15 s, and 72°C for 20 s. Results were analyzed with Rotor-Gene 6 and qBase quantification software ( http://medgen.ugent.be/qbase/). The data are expressed according to the delta-delta-Ct method according to com/cms/groups/mcb_support/documents/generaldocuments/cms_042380.pdf, and results represent one experiment out of at least two independent samplings, for which two preparations of cDNA were examined.

Analysis of Fruit MaMADS Box Gene Expression

Transcriptome analysis was performed on samples of fruits from four different developmental stages: harvest, prebreaker, breaker, and ripe. RNA was extracted as described above. Handling of samples for RNA sequencing has been described ( Fei et al., 2004), and levels of expression are expressed as RPKM. From these data, we selected, for further expression analysis, MaMADS box genes with high homology to known genes in tomato ( Solanum lycopersicum) affecting ripening, which also show expression above 50 RPKM in the pulp.

Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: GSMUSA Achr10P21480, MaMADS9; GSMUSA Achr5P16870, MaMADS5 (MuMADS1); GSMUSA Achr10P10990, MaMADS7; GSMUSA Achr2P13650, MaMADS10; GSMUSA Achr10P18600, MaMADS11; GSMUSA Achr11P19540, MaMADS1; GSMUSA Achr9P01370, MaMADS2; GSMUSA Achr9P29310, MaMADS8; GSMUSA Achr6P06040, MaMADS4; GSMUSA Achr2T10250, MaMADS3; Solyc07g055920, SlTAGL1; Solyc11g028020, SlTAGL11; Solyc03g114830, SlFUL2; Solyc06g069430, SlFUL1; Solyc05g012020, SlRIN.

Supplemental Data

The following supplemental materials are available.

View Abstract

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Banana MaMADS Transcription Factors Are Necessary for Fruit Ripening and Molecular Tools to Promote Shelf-Life and Food Security

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Genetic solutions to postharvest crop loss can reduce cost and energy inputs while increasing food security, especially for banana ( Musa acuminata ), which is a significant component of worldwide food commerce. We have functionally characterized two banana E class ( SEPALLATA3 [SEP3]) MADS box genes, MaMADS1 and MaMADS2 , homologous to the tomato ( Solanum lycopersicum ) RIN-MADS ripening gene.

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The SEP4 Gene of Arabidopsis thaliana Functions in Floral Organ and Meristem Identity

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The ABC model of flower organ identity is widely recognized as providing a framework for understanding the specification of flower organs in diverse p...

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Presenters:

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Background

Plant MADS box proteins play important roles in a plethora of developmental processes. In order to regulate specific sets of target genes, MADS box proteins dimerize and are thought to assemble into multimeric complexes. In this study a large-scale yeast three-hybrid screen is utilized to provide insight into the higher-order complex formation capacity of the Arabidopsis MADS box family. SEPALLATA3 (SEP3) has been shown to mediate complex formation and, therefore, special attention is paid to this factor in this study.

Results

In total, 106 multimeric complexes were identified; in more than half of these at least one SEP protein was present. Besides the known complexes involved in determining floral organ identity, various complexes consisting of combinations of proteins known to play a role in floral organ identity specification, and flowering time determination were discovered. The capacity to form this latter type of complex suggests that homeotic factors play essential roles in down-regulation of the MADS box genes involved in floral timing in the flower via negative auto-regulatory loops. Furthermore, various novel complexes were identified that may be important for the direct regulation of the floral transition process. A subsequent detailed analysis of the APETALA3, PISTILLATA, and SEP3 proteins in living plant cells suggests the formation of a multimeric complex in vivo.

Conclusions

Overall, these results provide strong indications that higher-order complex formation is a general and essential molecular mechanism for plant MADS box protein functioning and attribute a pivotal role to the SEP3 'glue' protein in mediating multimerization.

Additional Data File

Floral Organ

Yellow Fluorescent Protein

Cyan Fluorescent Protein

Floral Organ Identity

Since the isolation of the first plant MADS box transcription factor gene, substantial knowledge has been gained about the biological functions of these developmental regulators in various plant species. A thorough analysis of the complete genome sequence from the model species Arabidopsis thaliana revealed the presence of 107 different members belonging to this transcription factor family, with known or predicted functions in floral induction, plant architecture, female gametophyte development, fruit formation, fruit ripening, pod shattering, nitrate signaling and floral organ development [-]. Already in the early 1990s, genetic studies using floral organ mutants in Arabidopsis and Antirrhinum majus, representing mutations in mainly MADS box transcription factor genes, led to the establishment of the robust 'ABC model' for floral organ formation []. According to this original model, organ identities are determined by combinations of three functions, in which the A-function is essential for the specification of sepal identity, A- and B-functions for petals, B- and C-functions determine stamen identity, and the C-function on its own is responsible for carpel formation. In Arabidopsis the A-function is defined by APETALA1 ( AP1) and APETALA2 ( AP2), the B-function by APETALA3 ( AP3) and PISTILLATA ( PI), and the C-function by AGAMOUS ( AG), from which only the AP2 gene does not belong to the MADS box family.

Although the original 'ABC model' describes well the homeotic mutations in the various floral mutants, the lack of floral organ formation outside the flower when B- and/or C-function MADS box genes were ectopically expressed indicated that more factors are required for the floral organ identity functions [, ]. In Arabidopsis, the SEPALLATA ( SEP) MADS box genes appeared to be the missing co-factors and this new class of floral organ identity genes was termed E-function genes []. In line with the refined and extended 'ABC model', combinatorial over-expression of A-, B- and E-function genes results in conversion of leaves into petals, whereas constitutive expression of B-, C- and E-function genes gives rise to the formation of stamens instead of leaves [-]. Like for many MADS box genes, functional redundancy exists for the E-function genes, and only in the sep1 sep2 sep3 triple mutant were clear phenotypical alterations observed, namely the conversion of the second and third whorl organs into sepals and the development of a new inflorescence from the central region of the floral meristem []. Mutation of the fourth Arabidopsis SEP gene ( SEP4) in a sep1 sep2 sep3 background resulted in the production of leaves only [] and reveals an important function for SEP4 in sepal development. In addition, these latter observations give supporting evidence for Goethe's so-called 'big metamorphose', which proposes that a genetic program for the development of leaves is the basis for the formation of the flower, implying that floral organs can be regarded as modified leaves []. More detailed analyses of double and triple sep4, cauliflower ( cal), and ap1 mutants and genetic titration experiments for the sep mutations demonstrated that SEP4 also has a role in establishing floral meristem identity and petal, stamen and carpel development []. Furthermore, the genetic titration experiments for the sep mutations described by Ditta and colleagues [] showed dosage effects and redundancy for the SEP genes. Similar conclusions were drawn in relation to ovule development, in which the SEP genes act in a dose-dependent manner together with the C-function gene AG and the D-function genes SEEDSTICK ( STK), SHATTERPROOF1 ( SHP1) and SHATTERPROOF2 ( SHP2) [].

In conclusion, all these genetic data point towards a central role for the SEP genes in floral meristem and floral organ development. The importance of this class of genes for floral development has been put forward from an evolutionary point of view as well. Based on detailed phylogenetic studies and the fact that SEP like genes have been isolated from angiosperms only, Zahn and colleagues [] suggested that the SEP genes might be the basis for the origin of flowers.

An intriguing question arising from the ABC model is how all these different MADS box transcription factors co-operate together at the molecular level. Part of this question could be answered based on in vitro biochemical assays [] and yeast two-, three- and four-hybrid experiments that were performed over the past decade (among others [, , ]). The yeast experiments revealed binary interactions between specific A-, B-, C-, D-, and E-function MADS box proteins and, furthermore, they suggest the assembly into higher-order complexes consisting of 'ABC'-function MADS domain proteins and dimers. These results support the notion that MADS box proteins are active in a combinatorial manner and, accordingly, the 'Quartet model' has been proposed for MADS box transcription factor functioning []. In this model, a pivotal role has been attributed to the SEP proteins (E-function), which are present in almost all known higher-order complexes and, thus, can be regarded as the 'glue' proteins of floral organ development. Similar higher-order complexes have been identified for MADS box proteins of other species, such as Antirrhinum [], chrysanthemum [], petunia [-] and tomato [], demonstrating that these types of interactions are conserved among angiosperm species. Furthermore, it has been shown recently that the SEP3 protein on its own is able to form homotetramers in vitro []. Based on all these findings, it is acceptable to use the 'Quartet model' as the working model for MADS box transcription factor functioning, although hardly any evidence for direct physical higher-order complex formation between MADS proteins in plant cells has been found. Recently, it has been shown that the transient interaction between the petunia MADS box proteins FLORAL BINDING PROTEIN11 (FBP11) and FBP24 in protoplasts can be stabilized by adding the FBP2 protein, suggesting that a multimeric protein complex is formed in living plant cells []. Furthermore, gel filtration experiments with native protein extracts revealed that the FLOWERING LOCUS C (FLC) MADS box transcription factor is present in high molecular weight complexes []. In conclusion, MADS box proteins are able to multimerize in plant cells and are present in large complexes in vivo; however, the exact composition and stoichiometry of these complexes remains unknown.

In this study a large-scale yeast three-hybrid screen was performed to unravel the capacity and selectivity of higher-order complex formation for Arabidopsis MADS box transcription factors, with a special focus on the SEP proteins. In total, 106 ternary interactions were scored and in 78 cases at least one SEP protein appeared to be involved. The obtained results illustrate that higher-order complex formation is common among MADS proteins, and that this mechanism is employed by all subfamilies of the MADS box family. Based on available expression data for the MADS box genes that code for the interacting proteins, previous mutant analyses, and interaction studies in living plant cells, biological functions could be proposed for particular SEP3 complexes.

Large scale yeast three-hybrid analysis

After the discovery that A. majus MADS box proteins are able to form multimeric complexes in yeast [], a small number of additional ternary and quaternary complexes has been identified for MADS box proteins from various species. Currently, approximately 20 potential higher-order complexes involving Arabidopsis MADS box proteins have been reported [, , , ] (Table S1 in Additional data file 1). Remarkably, the vast majority of these complexes contains the SEP3 protein, which suggests that proteins of this sub-clade are important mediators of higher-order complex formation.

To get a better understanding about the capacity and specificity of complex formation for Arabidopsis MADS box proteins in general, and for the SEP3 protein in particular, a large scale yeast three-hybrid screening was performed. For this purpose all MADS box protein dimers that were identified in the comprehensive yeast two-hybrid screening [] were reconstituted in yeast strain PJ69-4 mating type A (Table S2 in Additional data file 1) by expressing one of the two dimerization partners as a fusion with the activation domain (AD) of the yeast GAL4 transcription factor, while the other protein was fused to a nuclear localization signal only []. Subsequently, these yeast clones were screened against the available collection of single MADS box proteins fused to the GAL4 binding domain (BD) in yeast strain PJ69-4 mating type Alpha [].

In total, 27,400 combinations (274 dimers × 100 single proteins) were tested for ternary complex formation and this screen yielded 47 positives (Table S3 in Additional data file 1). The results reveal a preference for ternary complex formation with proteins of the same sub-class of MADS box proteins; in general, type II proteins interact with other type II proteins and the same holds for members of the type I sub-class. Besides the 47 higher-order complexes that were identified in this screen, nine additional dimers were found that were missed in the large-scale yeast two-hybrid screening performed by De Folter and colleagues [] (Table S4 in Additional data file 1). Most likely, this difference is caused by the more mild selection criteria used for the yeast three-hybrid experiments. Although, many new triple combinations were found, the total number of ternary interactions was much lower than expected and, to our surprise, none of the known complexes was identified. The latter discrepancy could be explained to a large extent by technical limitations of the system: many combinations could not be tested for ternary complex formation, because the two proteins that were fused to GAL4-AD and -BD were already able to form a dimer that activated the yeast reporter genes even without the incorporation of the third protein in the complex. For instance, we could not observe the interaction between SEP3, STK (dimer 257 in Table S2 in Additional data file 1) and AG [], because GAL4-AD-SEP3 and GAL4-BD-AG are able to dimerize and activate the yeast reporter []. Furthermore, the presence of an intrinsic transcriptional AD in about 20% of the Arabidopsis MADS box proteins [], including the SEP1 and SEP3 proteins [], limited drastically the number of combinations that could be tested for ternary interactions due to auto-activation of the yeast reporters.

SEP3 ternary complex formation

One of the main goals of the large-scale yeast three-hybrid screening was to obtain a comprehensive picture of the potential of SEP proteins to mediate higher-order complex formation. However, this objective was hampered by the large number of dimers formed by these proteins and auto-activation of the yeast reporters by the SEP proteins. To overcome the latter problem we mapped the auto-activation domain in the SEP3 protein in order to remove this domain from the protein. This SEP member was chosen because genetic studies [, ], transactivation assays [], and yeast two-hybrid experiments [] have revealed that SEP3 is the most 'active' member of the SEP clade. To predict the presence of potential transcriptional activation domains, a search for motifs was performed with the software program DILIMOT on the full-length sequences of all MADS box proteins that gave auto-activation in yeast []. In this screen, a total of ten motifs was found, including the ones that were identified for the AP1 protein previously [], and almost all appeared to be located in the carboxy-terminal region of the MADS box proteins (Table S5 in Additional data file 1). This observation supports results from previous studies, where transcriptional activation capacity was often detected in the carboxy-terminal domain of plant MADS box proteins [, , , ]. Subsequent analyses revealed that the identified motifs are underrepresented in the sequences of MADS box proteins that do not give auto-activation in yeast. Based on this, a decision tree model could be designed using those motifs that discriminate between auto-activating and non-auto-activating MADS box sequences, providing additional evidence for their role in transcriptional activation (Table S5 in Additional data file 1). As control, DILIMOT was used again to search for eventual overrepresented motifs in the set of MADS box proteins that do not give auto-activation in yeast. This search did not reveal any motif, consistent with their lack of transcriptional activation. When using the predicted auto-activation motifs to scan all proteins from the Arabidopsis genome, we found that these motifs are over two-fold overrepresented in transcription factors compared to all proteins, and that this overrepresentation is even higher (over four-fold) when analyzing proteins with at least two of the motifs present (Table S5 in Additional data file 1). This result provides additional validation for the putative role of the motifs in transcription activation. Note that one does not expect all transcription factors to be auto-activating, and, in addition, not all auto-activating transcription factors need to contain the same motifs.

Figure illustrates the putative transcriptional activation motifs in the SEP3 protein sequence. Previous studies have demonstrated that besides transcriptional activation capacity, ternary interaction determinants are also localized in the carboxy-terminal region of MADS box proteins [], and, therefore, it was important to take this characteristic into account as well. Yang and Jack [] performed an in-depth mapping of the domains involved in ternary complex formation between the B-function proteins and SEP3, and this study assigned an important role to the last predicted amphipathic alpha-helical structure at the border between the K-box and the carboxy-terminal region (Figure ). Stimulated by these results, we used the web-based programs Paircoil [] and Multicoil [] to predict alpha-helical structures within the SEP3 protein. Based on these predictions and the identified putative activation domains, we designed two truncated SEP3 proteins lacking 80 and 67 amino acid residues at the carboxyl terminus, and named SEP3ΔC1 and SEP3ΔC2, respectively (Figure ). The first truncated protein stops within the last predicted alpha helix, while the SEP3ΔC2 protein terminates directly after this predicted structural domain. Subsequently, the shortened proteins were fused to GAL4-BD and tested in yeast for auto-activation capacity, which appeared to be abolished in both cases. To investigate the ability of the two truncated SEP3 versions to form dimers and higher-order complexes, the previously identified heterodimer between AG and SEP3 [] and the ternary complex between AG, STK and SEP3 [] were tested in yeast. As expected, both SEP3ΔC protein versions were still able to dimerize with AG; however, only SEP3ΔC2 interacted with AG and STK in the yeast three-hybrid experiment, demonstrating once more the importance of the predicted alpha-helical structure at the end of the K-box for ternary protein interactions (helix III in Figure ). Based on these observations, we reconstituted all known SEP3 dimers in yeast making use of the SEP3ΔC2 construct (Table S6 in Additional data file 1). This new collection of dimers was screened against all single MADS box proteins in a yeast three-hybrid assay, and reciprocally, the single SEP3ΔC2 protein fused to GAL4-BD was combined with the set of MADS domain dimers (Table S2 in Additional data file 1). This experiment yielded 59 additional higher-order complexes (Table S7 in Additional data file 1), including the known SEP3 ternary interactions (Table S1 in Additional data file 1). Figure shows the sub-network representing all SEP3 interactions, whereas the overall network, including the complexes listed in Table S3 in Additional data file 1, is depicted in Figure .

SEP3 complex partners are co-expressed

A prerequisite for a biologically relevant protein-protein interaction in planta is coexistence of the proteins in the same cell and at the same moment during development. Therefore, the expression patterns of the genes encoding complex-forming MADS box proteins were compared using AtGenExpress data []. Note that a few MADS box genes are not presented on the ATH1 arrays used for AtGenExpress. For these particular MADS box genes, the AtTAX data were analyzed. This data set represent the results from whole genome tiling array hybridizations []. Unfortunately, no expression above background levels could be detected for most of the MADS box genes missing from the ATH1 arrays in the limited number of tissues tested on the tiling arrays. As a consequence, co-expression could not be confirmed for 16 out of the 106 identified complexes. Except for one complex, these are all complexes involving type I MADS box proteins, which are hardly studied. The co-expression analysis revealed that for almost 100% of the identified complexes containing type II MADS box proteins, the encoding genes have an overlap in expression in at least one tissue (Tables S3 and S7 in Additional data file 1). Remarkably, for type I proteins this was only 78%. This may reflect a real lack of co-expression, but, more likely, this is due to the low and very localized expression of a number of type I proteins [, , -], which makes the microarray data less reliable. For the few identified complexes consisting of combinations of type I and type II proteins, the expression patterns of the encoding genes appeared to overlap. The high percentage of co-expression (overall 95%) indicates that almost all identified complexes could potentially be formed in planta, although, for some of the genes, the expression levels were very low in the overlapping tissues. We also realize that these data are mRNA expression data and do not reflect protein levels; however, as far as is known, the spatial and temporal distribution of MADS domain proteins follows roughly the mRNA expression patterns [, ]. Nevertheless, we can not exclude that non-cell autonomous action of MADS proteins plays a role and that some proteins are transported to adjacent cell layers and tissues. This has been shown, for instance, for the B-function MADS box proteins from Antirrhinum []. In Figure S1 in Additional data file 1 a comparison of expression patterns is presented for all gene combinations encoding putative ternary complex components for the complexes that contain the SEP3 protein.

SEP3, AP3, and PI complex formation in living plant cells

To our surprise, a ternary complex was found in yeast between AP3, PI and SEP3, making use of full-length B-function proteins (Table S7 in Additional data file 1). Previous experiments revealed that the supposed heterodimer between AP3 and PI could not be detected in the yeast two-hybrid system when full-length proteins were used [, ]. This strongly suggests that SEP3 can mediate the interaction between AP3 and PI in yeast. To investigate the behavior of these proteins in plant cells in more detail, we analyzed their interactions by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM) in Arabidopsis leaf cells [, , ]. Initially, AP3, PI and SEP3 were carboxy-terminally labeled by enhanced cyan fluorescent protein (CFP) or enhanced yellow fluorescent protein (YFP) and transiently expressed in protoplasts, followed by confocal laser scanning microscopy for the analysis of their intracellular localization. Surprisingly, besides SEP3, PI was also nuclear localized, whereas the AP3 protein was found in both the nucleus and cytoplasm (Figure ). These localization results are not in agreement with previous intracellular localization data obtained for AP3 and PI in studies by McGonigle and colleagues [], who observed that nuclear localization of the two B-function proteins occurs only when both proteins are simultaneously expressed. However, in their case, the GUS reporter was used and amino-terminally fused to the MADS box protein, followed by expression in onion epidermal cells, which might be the reason for the observed differences. It has been shown before that fusion of green fluorescent protein-like fluorophores to the amino terminus of MADS box proteins can influence their nuclear import [, ]. To analyze whether there is a difference between amino- and carboxy-terminal labeling with respect to localization, AP3 and PI were also labeled with YFP at the amino terminus and transfected into protoplasts. In accordance with the results reported in the literature [], most of the signal appeared to be localized in the cytoplasm in this case (Figure ); however, co-expression of the other B-function protein labeled at the carboxy-terminal results in a mainly nuclear localized signal for both proteins (Figure ) and the same result was obtained when both proteins were carboxy-terminally labeled (Figure ). Based on these observations, we decided to make use of carboxy-terminal fusions for all further experiments.

FRET-FLIM was used to investigate the physical interaction of the labeled proteins in the leaf cells. The homodimer combinations 'SEP3-CFP + SEP3-YFP', 'PI-CFP + PI-YFP' and 'AP3-CFP + AP3-YFP' were analyzed first and 'PI-YFP + free CFP' was used as a negative control (Figure ). Interestingly, a remarkable difference was detected among the proteins analyzed for homodimerization capacity. In the case of SEP3, a strong reduction of the fluorescence lifetime was observed over the entire nucleus, suggesting efficient homodimer formation (Figure ). In contrast, AP3 and PI showed only a strong reduction of fluorescence lifetime in particular sub-nuclear spots, which may represent more transient interactions (Figure ). Interaction in parts of the nucleus has been reported before for petunia MADS box proteins []. Currently, it is unclear whether these non-homogeneous interactions are biologically relevant; however, the ability of B-function proteins to homodimerize is supposed to be the ancestral status, which subsequently evolved into obligatory heterodimerization in the core eudicots []. In line with this, it could be that the homodimer interactions identified for the individual Arabidopsis B-function proteins by FRET-FLIM are remnants of their former ability to homodimerize, which has been almost lost during evolution. In a following experiment, we tested the supposed heterodimerization between the full-length PI and AP3 proteins in plant cells. Because no interaction was found between these two full-length proteins in yeast, the heterodimer between AP1 and SEP3 was added as a positive control []. As expected, the AP1-SEP3 combination showed a very strong reduction in fluorescence lifetime over the entire nucleus (Figure ). Interestingly, the combination AP3-PI also showed a strong FRET-FLIM signal demonstrated by a short fluorescence lifetime, suggesting that these proteins are able to form heterodimers in living plant cells (Figure ). Remarkably, this combination always resulted in a strong accumulation of fluorescent signal in a ring-like pattern at the position of the nucleolus (Figures and ), a phenomenon that was never observed for any other combination of MADS box proteins tested.

Subsequently, the effect of SEP3 on the AP3-PI heterodimer was analyzed by FRET-FLIM to gain insight into higher-order complex formation. For this purpose the occurrence of FRET was measured between PI-CFP and AP3-YFP in the presence of a non-labeled SEP3 protein. The addition of SEP3 appeared to have a strong effect on the localization of the PI and AP3 proteins: instead of localization at the nucleolus (Figure ), the AP3 and PI protein interaction appeared to be more equally distributed over the nucleus in the presence of SEP3 (Figure ). Furthermore, a short fluorescence lifetime could be observed over the entire nucleus, although the drop in fluorescence lifetime was less strong than in the absence of SEP3 (Figure ). An explanation for this could be that SEP3, which is supposed to bind to the carboxy-terminal regions of AP3 and PI, interferes with the optimal positioning of CFP and YFP for a high FRET efficiency.

Plant MADS domain protein higher-order complex formation

MADS box transcription factors play essential roles during the plant lifecycle and can be characterized as the architects of plant development. Their specific functioning is mainly determined by direct physical protein-DNA and protein-protein interactions (reviewed in [, ]). Besides the formation of dimers, the well studied type II floral organ identity MADS box proteins [] are supposed to form multimeric protein complexes consisting of three to four different MADS box proteins (for example, [, , ]). Remarkably, the majority of higher-order complexes known to date contains at least one protein belonging to the 'E-function' class, which is represented by the SEP proteins in Arabidopsis []. It was unknown whether assembly into these large complexes is a common molecular mechanism that mediates plant MADS box transcription factor functioning, or whether this is only characteristic for the 'ABC-function' proteins and, in particular, for 'E-function' proteins. Therefore, we performed a large-scale yeast three-hybrid analysis for members of the Arabidopsis MADS box transcription factor family. Although this study was not comprehensive due to technical limitations of the screen, many novel complexes could be identified for both type I and type II MADS box transcription factors. In the initial screen with the full-length proteins, more complexes were identified that exclusively consist of type II proteins (25) than complexes with only type I proteins (15), while the Arabidopsis genome encodes more proteins belonging to the latter class. Whether this difference in the capacity to assemble into multimeric complexes between these two groups is due to differences in protein structure and reflects their biological functions needs more thorough investigations by alternative methods. The fact that type I proteins lack a K-box, which has been shown to be an important mediator for dimerization and higher-order complex formation [, ], could explain the observed differences. Nevertheless, coiled-coil structures have been predicted within the carboxy-terminal region of type I proteins [] and these structural motifs are well-known molecular recognition structures [] that potentially can be involved in type I complex formation.

In the previous two-hybrid screen from De Folter and colleagues [], interactions between type I and type II MADS box proteins were observed, although rare. In the current three-hybrid screen also only a few complexes (7) were found that contain both type I and type II proteins, though the genes encoding these interacting proteins are co-expressed (Table S3 in Additional data file 1). The presence of these interactions suggests that they arose before the duplication that gave rise to the two lineages, which happened before the divergence of plants and animals []. Alternatively but less likely, these hybrid interactions were acquired after the birth of the type I and II MADS box lineages. Interestingly, the interaction networks of the type I and type II proteins are clearly separated (Figure ), which may reflect the different functions these proteins play in plants. Most type II proteins are involved in identity specification and phase changes, while recent studies on type I genes [, , -] support the notion that they play an important role in gametophyte and embryo development. The inter-lineage interactions between the type I and II sub-networks may link the different roles these MADS box proteins play. In this respect it is interesting to notice that five out of seven 'type I-type II' interactions contain either the type II proteins ARABIDOPSIS BSISTER (ABS) or AG; both proteins are important for gametophyte and seed development in Arabidopsis [, , ]. The ABS gene encodes two proteins, ABS-I and ABS-II, which are derived through alternative splicing []. The yeast three-hybrid experiments revealed that both proteins multimerize with type I proteins, but with a difference in specificity. Besides these differences, novel and distinctive interactions with type II proteins were also found for the two ABS proteins, which had not been identified in previous studies [, ]. These differences in interaction specificity probably explain the observation that only the long splice form (ABS-I) can complement the endothelium defects in the abs mutant []. In contrast to ABS-II, the ABS-I protein is able to form a ternary complex with AGAMOUS-LIKE16 (AGL16)-SEP3, PI-SEP3, AGL74N-SEP2 and SEP1-SEP2. Except for 'AGL74N-SEP2-ABS-I', co-expression of the genes encoding these interacting proteins in carpels and young pistils containing seeds has been detected []. Unfortunately, detailed information about expression in the ovule and function of these ABS-I specific interaction partners is missing, leaving the question of whether one of these novel complexes is responsible for the functional discrepancy between ABS-I and ABS-II unanswered.

Expression of the genes encoding complex members

In general, co-expression of genes encoding interaction partners may give clues about a common function for the proteins involved. For example, members of the MIKC* sub-clade (also known as Mδ []) are specifically expressed during pollen formation and the encoded proteins form higher-order complexes with other members of this sub-clade, suggesting that they play an important role during pollen development []. However, a lack of a large expression overlap in planta does not necessarily mean that we are dealing with a false positive protein interaction. Note that, for example, the AG-SEP3 dimer interacts with a set of ternary interacting factors that overlap in expression pattern with the dimerization partners in distinct tissues, or during particular stages of development only (Tables S3 and S7 and Figure S1 in Additional data file 1). Complexes were also identified for proteins that show no obvious overlap in their corresponding mRNA expression patterns, as, for example, complexes consisting of the floral activators AGL24 [], SUPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) [], and the AGL17 or AGL19 proteins, which are both encoded by genes preferentially expressed in roots [, ]. However, recent functional analyses of AGL17 [] and AGL19 [] revealed that these proteins are also inducers of flowering and share this function with their putative complex partners. Besides the expression in roots, both AGL17 and AGL19 show low expression in above-ground vegetative parts [, ], which probably results in sufficient molecules for complex formation and subsequent activation of flowering in the shoot apical meristem. Furthermore, it is known that the expression levels of AGL24 [], SOC1 [], and AGL17 [] are coordinately up-regulated by CONSTANS (CO) and, hence, that these MADS box genes act downstream of this protein in the photoperiodic flowering pathway. Based on all these findings, we hypothesize that the specific higher-order complex formation between these MADS box proteins is an important mechanism for the functioning of these proteins in the regulation of flowering time. Notably, similar kinds of complexes have been found for a couple of other related and preferentially root-expressed MADS box proteins (AGL14, AGL21 and AGL42) [, , ], whose functions are unknown. From the genes encoding these proteins, AGL42 is strongly up-regulated upon a switch from short day to long day conditions, as is the case for SOC1 and AGL24 []. Based on the common complex formation partners identified in this study, we may speculate that the AGL42 protein also plays a role in floral induction.

The importance of SEP proteins for multimerization

SEP proteins seem to be important mediators of higher-order complex formation and, therefore, we have focused on the capacity of the SEP3 protein to form multimeric complexes. In the dedicated yeast three-hybrid screen with the carboxy-terminally truncated SEP3 protein, known SEP3 ternary complexes were confirmed, showing that the conditions of our yeast three-hybrid assay permit the detection of these ternary interactions. To our surprise, the screen with the truncated SEP3 protein more than doubled the total number of identified ternary MADS box protein complexes. Despite the fact that the number of ternary interactions found in this study resembles most likely only a small proportion of the potential higher-order complexes present in Arabidopsis, this result reveals an important role for SEP3 in MADS box protein complex formation. Therefore, the SEP3 protein can be regarded as a 'glue' that mediates the assembly of MADS box transcription factor complexes and is functional as a hub in the MADS box transcription factor interaction network. We may hypothesize that the other SEP proteins have a similar specificity for higher-order complex formation, knowing that there is functional redundancy within this clade of MADS box proteins [, ]. In line with this idea, the comprehensive yeast two-hybrid screening performed by us showed similar binary interactions for SEP1 and SEP3 []. However, SEP2 and SEP4-I/II seem to have a number of different dimerization partners in yeast; also in the yeast three-hybrid screen presented in this report, specific complexes were identified for SEP2 and SEP4-II that could not be found for SEP3. Together, this suggests that the functional redundancy present in the Arabidopsis SEP clade is not complete and, hence, that some of the SEP proteins have gained or maintained specific interactions and functions that are not shared by the other members of the family. A similar comprehensive approach as followed in this study for SEP3, consisting of mapping the auto-activation domain and performing the three-hybrid screen with mutated or truncated clones, would be needed for each individual SEP protein to elucidate their specific ternary complex formation capacities. Regardless of the outcome of such an experiment, however, it is clear from the genetic studies that besides small differences, there is overlap between the functions of the SEP proteins in the inner three whorls of the flower, which means that the different SEP proteins should have the capacity to form complexes with at least some common MADS box partners. Assuming that SEP3 is the 'glue' for higher-order complex formation in the inner three floral whorls, the question arises as to which SEP protein functions as 'glue' during the vegetative stage of development. SEP4 is expressed early during development in the green parts of the plant, in contrast to SEP3 [], though at relatively low levels. Because of this, it may also be possible that another type II MADS box protein is functional as a 'glue' protein during the vegetative stage. In this respect, SOC1 is a good candidate, because it has the right spatial expression pattern and a large number of two-hybrid interaction partners like the SEP proteins. It functions as a hub in the two-hybrid network [] and, more importantly, this protein is incorporated in ternary complexes almost as frequently as SEP3 (Tables S3 and S7 in Additional data file 1).

Biological functions of ternary SEP3 MADS box protein complexes

Studies performed previously revealed the importance of SEP proteins present in ternary and quaternary floral organ identity complexes [, ] and recent in planta protein localization studies showed co-localization of the 'ABC' proteins in accordance with the 'ABC model' []. Besides these interactions with other ABC-function MADS box proteins, our results have shown that the SEP3 protein is potentially incorporated in complexes with MADS box proteins involved in the regulation of flowering time, such as SOC1 [], AGL24 [], SHORT VEGETATIVE PHASE (SVP) [], and AGL15 [] (Figure ). These interactions suggest that the SEP3 protein also functions in the transition to flowering, which is in line with observations in a study by Pelaz and colleagues [], who obtained an enhanced early flowering phenotype for Arabidopsis plants ectopically expressing both AP1 and SEP3 when compared to plants over-expressing AP1 alone. Expression of the SEP3 protein could not be detected in vegetative tissues; however, the protein is present at low levels in the inflorescence meristem []. SEP3 probably performs this early function redundantly with SEP4, which, in contrast to SEP3, is expressed during the vegetative stage of development and is also able to form a couple of ternary complexes with the flowering time MADS box proteins. In addition or alternatively, ternary complexes consisting of MADS box proteins involved in regulation of flowering time and floral organ identity proteins (for example, SEP3, AG, AP1) could function in negative auto-regulatory feed-back mechanisms (Figure ). De Folter and colleagues [] hypothesized that the expression of genes encoding floral inducing MADS box proteins is down-regulated in the floral organ primordia by a negative auto-regulatory loop involving dimerization of the encoded proteins with the MADS box proteins functioning in floral organ development []. Recently, the research group of Yu showed that the floral meristem identity protein AP1 is involved in the down-regulation of the flowering time genes SOC1, SVP and AGL24 []. Based on our results, it is tempting to speculate that down-regulation of these flowering time genes is mediated by a negative feed-back loop, in which both the flowering proteins and SEP3 are involved (Figure ). In line with this, in situ hybridization analyses for flowering time genes in wild-type plants show hardly any signal during later stages of flower development [, ]. However, in mutant backgrounds of floral organ identity MADS box genes, such as ap1, ag, and sep1/ sep2/ sep3, ectopic expression of these flowering time genes is obtained in floral tissues [, ]. Although this gives strong evidence for the supposed negative auto-regulatory loops, further studies are required to support the hypothesis that higher-order complexes are essential for this function.

It is difficult to assign a biological role for some of the other ternary SEP3 complexes identified in our study because no information is available about the functions of the individual proteins. Furthermore, many proteins may have multiple functions throughout the life cycle of a plant and, therefore, late functions can be masked by early functions in genetic studies. The expression of MADS box genes late during development of the floral organs [] and the late functions identified for, for example, B-function MADS box proteins [, ] and AG [] demonstrate that these transcription factors are multi-tasking and play a role during further differentiation of the floral organs. These various functions are reflected in the different complexes formed by such a MADS box protein, each supposed to regulate a specific set of target genes. SEP3 is part of many complexes and, therefore, may bind to different target genes controlling distinct developmental pathways. Because the sep1 sep2 sep3 triple mutant produces only sepals in the flower [], it is difficult to predict SEP3 functions at later flowering stages, but, based on its expression pattern and our interaction data, we could assign a role for this protein as a key regulator in many developmental processes (Figure ). For instance, the protein complex consisting of SEP3, STK and AG is involved in ovule identity specification [], while a combination with the integument and seed coat-specific protein ABS may be required for the subsequent steps in seed development [].

Molecular function of SEP3 in ternary MADS box protein complexes

As shown by yeast-based and FRET-FLIM studies, the ternary factor SEP3 is able to stabilize dimeric interactions and to affect the subcellular localization of its interaction partners. Stabilization of a MADS box transcription factor dimer by a ternary factor has been shown in petunia before [] and may be a general function for ternary MADS box factors. The effect of SEP3 on AP3-PI localization could play an important role in the temporal storage or, alternatively, in the activation of this specific MADS box protein dimer. Recently, it has been shown that the mammalian basic helix-loop-helix transcription factor Hand1 is sequestered in the nucleolus due to interaction with a co-factor, and that the release of this protein from the nucleolus is essential for its activation []. Similar mechanisms may play a role in activating particular plant MADS box proteins, such as AP3 and PI. The question remains if this is the only function for ternary factors, like SEP3, in higher-order complexes. SEP3 appears to contain a strong transcription activation domain and, based on this, it has been hypothesized that an important function of this ternary factor is to add transcriptional activity to multimeric transcription factor complexes []. This might be true, but, at least in the case of the complex AP3-PI-SEP3, the SEP3 protein is doing more, because plants with constitutive overexpression of AP3 and PI fused to the VP16 trans-activation domain do not show homeotic changes of cauline leaves into floral organs (K Goto, personal communication). In contrast, the constitutive expression of AP3 and PI in combination with SEP3 gives conversions of cauline leaves into petals [, ]. Although the combination of constitutive AP3 and PI-VP16 expression is sufficient to activate the positive auto-regulatory loop for the B-function MADS box genes - that is, it activates the AP3 promoter [] - it is not sufficient for the regulation of all AP3-PI target genes that are essential for petal development. In conclusion, SEP3 can change the sub-nuclear localization of the AP3-PI heterodimer and probably this is crucial for petal development. Furthermore, Egea-Cortines and colleagues [] have shown that ternary complexes bind more strongly to the consensus CArG-box in DNA sequences than MADS box protein dimers. SEP3 in a multimeric complex may facilitate the protein-DNA interaction, either by stabilizing the dimer or by direct binding to the DNA and providing specificity. In the latter case, the DNA will bend and the transcription complex will bind to two binding sites at a short distance from each other. In addition, ternary complex formation may play a role in the recruitment of co-factors. Recent studies have shown that the MADS box proteins AGL24 and SVP are not able to interact with the LEUNIG-SEUSS co-repressor complex, although interaction between these proteins could be mediated by the AP1 protein []. In a similar way, SEP3 enables the interaction between MADS box proteins involved in ovule development (for example, STK) and the BELL1 homeodomain factor [].

This study yielded a collection of potential multimeric MADS domain protein complexes in which SEP3, the 'glue protein', plays a central role. Besides the initial steps of floral organ formation, this protein seems to function in various other plant developmental processes via multimerization (Figures and ). Higher-order complex formation of MADS domain proteins appears to be a common process and provides these transcription factors with unique attributes to function in a specific manner, such as the possibility to change interaction stability, localization of the proteins, and their DNA binding specificity. Combining protein interaction analyses as performed in this study and co-expression analyses provides complementary functional information about MADS transcription factors, in particular when mutant phenotypes are missing due to redundancy or when the proteins are involved in multiple developmental processes, as is the case for SEP3.

Plant material

Protoplasts were obtained from Arabidopsis thaliana Col-0 leaves, which were grown under normal greenhouse conditions (16/8 h light/dark, 22°C), according to Aker and colleagues [].

Plasmid constructions

For the yeast three-hybrid experiments two new SEP3 Gateway entry clones were generated, encoding the carboxy-terminally truncated versions of this protein. The first clone, designated SEP3ΔC1, encodes SEP3 lacking the last 80 amino acids of the carboxyl terminus and the second clone, SEP3ΔC2, encodes the SEP3 protein lacking 67 amino acids at its carboxyl end. The truncated coding regions were obtained by PCR and a new stop codon was included in the reverse primer. Subsequently, the PCR fragments were cloned into pCR8/GW/TOPO (Invitrogen, Carlsbad, CA, USA), followed by sub-cloning via a Gateway LR reaction into pBDGAL4 (pDEST32; Invitrogen) and the Gateway compatible pTFT1 yeast expression vector (pARC352) []. For the in vivo localization and interaction studies, the coding region of the MADS box genes APETALA1 ( AP1), APETALA3 ( AP3), PISTILLATA ( PI), AGAMOUS ( AG), SEP3 and SEP 3ΔC1, were cloned as Gateway entry clones without stop codons (pCR8/GW/TOPO; Invitrogen), in order to allow carboxy-terminal fusions. The obtained entry clones were recombined into the Gateway compatible destination vectors pARC971 and pARC428 from which expression is driven by the constitutive CaMV35S promoter and that contain the coding regions of the fluorophores CFP and YFP, respectively []. Furthermore, amino-terminal fusions were made for AP3 and PI. In this case, the destination vector was pK7WGY2,0 from the VIB collection [], containing the coding region of the YFP molecule. AP3 and PI entry clones including stop codons were taken from the REGIA collection [, ]. All plasmids were controlled by sequence analyses (DETT sequence kit; Amersham, Sunnyvale, CA, USA).

Yeast three-hybrid screen

Transformations of yeast strain PJ69-4, mating type A and α [], were done as described by the laboratory of Gietz []. Triple combinations of MADS box proteins in yeast were obtained by robotized mating between individual matα yeast cultures containing 'pBD-GAL4-MADS' vectors [] and matA yeast cultures containing the MADS dimers (Tables S2 and S6 in Additional data file 1), following the protocol described before []. The mated yeast was grown for 2 days at 30°C on plates with synthetic dropout medium without leucine (L), tryptophan (W) and adenine (A), to select for the presence of all three plasmids. Subsequently, some yeast material was resuspended in 50 μl sterile water, in a 96-well micro-titer plate. Aliquots (5 μl) of these suspensions were spotted onto synthetic dropout medium plates lacking the amino acids L, W, A and histidine (H) and supplemented with 1 mM 3-amino-1,2,4-triazole (3-AT) in a grid of 96 spots, to select for protein interactions. These plates were incubated at 20°C for 7 days before scoring of yeast growth. All positives due to dimerization between two of the three proteins, and/or auto-activation by the MADS box protein expressed from the pBD-GAL4 vector, or its dimerization partner in the pARC352 vector, were discarded based on our knowledge from the large-scale yeast two-hybrid experiment []. For all remaining positive combinations the mating was repeated and the interaction confirmed by spotting onto plates with synthetic dropout medium lacking amino acids L, W, A, H and supplemented with 1, 5, or 10 mM 3-AT. In parallel, a LacZ assay [] was performed to test for the activation of this second reporter gene. All combinations that were scored at least two times positive and for both the HIS and LacZ selection markers, were selected as true positives.

Fluorescence microscopy in living cells

Arabidopsis leaf protoplasts were transfected as described by Aker and colleagues []. Plasmid DNA (15-30 μg) was used and the protoplasts were incubated overnight at 25°C before imaging. Images were made using a confocal laser microscope 510 (Carl Zeiss, Jena, Germany). The Argon laser was used to excite at 458 and 514 nm for CFP and YFP, respectively. Fluorescence was detected through a band pass filter of 470-500 nm for CFP and 535-590 nm for YFP [].

FRET-FLIM measurements in living cells

FRET-FLIM analyses were done in Arabidopsis protoplasts as described before [, ]. The donor fluorescence lifetime was measured on the central part of the nucleus of each single cell, pixel by pixel, and at least ten cells were analyzed per combination in three independent experiments. The donor lifetime of CFP was fixed at 2.6 ns for all further analyses. Images were acquired by using the Becker and Hickl 1 SPC 830 module, and SPC image 2.8 software was used for the data analyses (Becker and Hickl, Berlin, Germany).

Prediction of transcriptional activation

DILIMOT [] was applied using default parameters (maximum motif length 8, number of fixed positions 3, minimal number of motifs in dataset 3) on all 19 sequences of MADS box proteins showing auto-activation in yeast []. Subsequently, using ps_scan [], it was confirmed that these motifs occur much less often in other MADS box protein sequences. To obtain further insight into the role of these motifs, a decision tree model was built (using the function 'tree' in the software package R) with class indicator 'auto-activation' or 'no auto-activation' and variables describing occurrence of each motif in the sequences. This analysis selected five motifs out of the ten motifs returned by DILIMOT (Table S5 in Additional data file 1) and resulted in a model with over 80% accuracy, 80% specificity and 50% coverage. The accuracy is the overall percentage of correct predictions and the specificity indicates the percentage of predicted auto-activating proteins for which auto-activation was identified in yeast. The coverage gives the percentage of experimentally detected auto-activating proteins that were also predicted to give auto-activation.

Co-expression analysis

The developmental set of the AtGenExpress expression atlas [] was analyzed for expression of MADS box genes, as previously described []. A threshold of log ₂ ≥ 4 was applied to identify overlap in tissues with expression of genes. For genes not expressed in the AtGenExpress expression atlas ( AGL13, AGL61, AGL92, AGL96, and AGL103) other publicly available expression data were used [, , ].

The following additional data are available with the online version of this paper. Additional data file contains Tables S1-S7 and Figure S1. Table S1 lists all higher-order complexes for Arabidopsis MADS domain proteins reported in the literature. Table S2 provides an overview of all MADS domain protein dimers that have been generated in yeast. Table S3 presents the higher-order complexes that were identified in the initial yeast three-hybrid screening. Table S4 lists the few dimers that could be extracted from the dataset in Additional file 3 and that were not identified before in the comprehensive two-hybrid screening []. Table S5 gives information about the prediction of transcription activation domains in the MADS protein sequences. Table S6 lists the yeast collection containing all SEP3ΔC2 dimers. Table S7 lists all ternary complexes that were identified using this SEP3ΔC2 collection. Figure S1 shows the data of the co-expression analysis for genes encoding interacting MADS domain proteins.

Richard GH Immink, Isabella AN Tonaco contributed equally to this work.

Acknowledgements

We would like to thank Paul Mooyman for technical assistance. The original pTFT1 yeast expression vector was kindly provided by Dr Hans Sommer. We are grateful to K Goto for sharing unpublished results with us and to the anonymous reviewers, who provided useful comments and suggestions to improve the manuscript. This work has been financially supported by CAPES-Brazil (BEX 0970/01-8), the Netherlands Proteomics Centre (NPC) and a NWO-VENI grant to RGHI.

Electronic supplementary material

Authors' contributions

RGHI supervised the projects in which the work was carried out, performed part of the cloning, set-up the yeast three-hybrid screening method, and wrote the manuscript together with INT. In addition, INT performed the localization and FRET-FLIM experiments. SdF performed the network and co-expression analyses and was involved in scientific discussions. All yeast vectors and the yeast collections and glycerol stocks were prepared by AS. ADJvD performed the bioinformatics predictions for transcription activation domains and JBL performed the yeast three-hybrid screenings. The FRET-FLIM experiments were supervised by JWB and he performed a few experiments of this type. GCA contributed to all scientific discussions and critically revised the manuscript.

Below are the links to the authors' original submitted files for images.

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SEPALLATA3: the 'glue' for MADS box transcription factor complex formation

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Plant MADS box proteins play important roles in a plethora of developmental processes. In order to regulate specific sets of target genes, MADS box proteins dimerize and are thought to assemble into multimeric complexes. In this study a large-scale yeast three-hybrid screen is utilized to provide insight into the higher-order complex formation capacity of the Arabidopsis MADS box family.

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Abstract

During reproductive development, rice plants develop unique flower organs which determine the final grain yield. OsMADS1, one of SEPALLATA-like MADS-box genes, has been unraveled to play critical roles in rice floral organ identity specification and floral meristem determinacy. However, the molecular mechanisms underlying interactions of OsMADS1 with other floral homeotic genes in regulating flower development remains largely elusive. In this work, we studied the genetic interactions of OsMADS1 with B-, C-, and D-class genes along with physical interactions among their proteins. We show that the physical and genetic interactions between OsMADS1 and OsMADS3 are essential for floral meristem activity maintenance and organ identity specification; while OsMADS1 physically and genetically interacts with OsMADS58 in regulating floral meristem determinacy and suppressing spikelet meristem reversion. We provided important genetic evidence to support the neofunctionalization of two rice C-class genes ( OsMADS3 and OsMADS58) during flower development. Gene expression profiling and quantitative RT-PCR analyses further revealed that OsMADS1 affects the expression of many genes involved in floral identity and hormone signaling, and chromatin immunoprecipitation (ChIP)-PCR assay further demonstrated that OsMADS17 is a direct target gene of OsMADS1. Taken together, these results reveal that OsMADS1 has diversified regulatory functions in specifying rice floral organ and meristem identity, probably through its genetic and physical interactions with different floral homeotic regulators.

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Interactions of OsMADS1 with Floral Homeotic Genes in Rice Flower Development

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During reproductive development, rice plants develop unique flower organs which determine the final grain yield. OsMADS1, one of SEPALLATA-like MADS-b...

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Abstract

The MADS transcription factors (TF) constitute an ancient family of TF found in all eukaryotes that bind DNA as obligate dimers. Plants have dramatically expanded the functional diversity of the MADS family during evolution by adding protein-protein interaction domains to the core DNA-binding domain, allowing the formation of heterotetrameric complexes. Tetramerization of plant MADS TFs is believed to play a central role in the evolution of higher plants by acting as one of the main determinants of flower formation and floral organ specification. The MADS TF, SEPALLATA3 (SEP3), functions as a central protein-protein interaction hub, driving tetramerization with other MADS TFs. Here, we use a SEP3 splice variant, SEP3 Δtet, which has dramatically abrogated tetramerization capacity to decouple SEP3 tetramerization and DNA-binding activities. We unexpectedly demonstrate that SEP3 heterotetramer formation is required for correct termination of the floral meristem, but plays a lesser role in floral organogenesis. The heterotetramer formed by SEP3 and the MADS protein, AGAMOUS, is necessary to activate two target genes, KNUCKLES and CRABSCLAW, which are required for meristem determinacy. These studies reveal unique and highly specific roles of tetramerization in flower development and suggest tetramerization may be required to activate only a subset of target genes in closed chromatin regions.

INTRODUCTION

Plants maintain a pool of pluripotent stem cells that give rise to new organs throughout their lifecycles. These stem cells are located at the root and shoot apical meristems from which all below ground and aerial tissues derive. During angiosperm reproduction, the shoot apical meristem is converted to an inflorescence meristem (IM) and finally to fully determinate floral meristems (FM), which terminate pluripotent cell production and commit meristematic cells to distinctive fates. From the FM, four types of floral organs are produced in whorls with the sepals, petals, stamen and carpel of the flower located in the outer-most to the inner-most whorls of the flower. The MADS family plays critical roles in all aspects of flowering, including the floral transition, floral meristem determinacy and floral organ development (for recent reviews ( 1, 2)). One of the best-studied developmental networks involves the ABCE class MADS genes whose overlapping expression patterns control floral meristem identity, determinacy and the identity of the floral organs.

In Arabidopsis, the A gene, APETALA1 ( AP1), is required for floral meristem identity, sepal and petal formation, the B genes, APETALA3 ( AP3) and PISTILLATA ( PI), are required for petal and stamen development, the C gene, AGAMOUS ( AG), is required for determinacy of the floral meristem as well as male and female organogenesis and the E class genes, SEPALLATA1, SEPALLATA2, SEPALLATA3 and SEPALLATA4 ( SEP1-4), are necessary for floral organ identity in all whorls ( 3-6). Single knock outs of any of the SEP genes do not have strong floral phenotypes, however in the sep1 sep2 sep3 (sep1/2/3) triple mutant all floral organs are converted to sepaloid-like structures with a lack of determinacy of the floral meristem resulting in the continuous generation of sepals where the carpel would exist in wild type (WT) flowers ( 3). Initial expression patterns of the class B and C genes in the triple mutant are unaffected, underscoring the critical role of the SEP genes in both floral organ formation and floral meristem identity. The sep1/2/3/4 quadruple mutant is further affected, with floral organogenesis abolished, resulting in leaf-like organs in all whorls and a reduction in the expression levels of the B and C class genes ( 7, 8).

The A-E class MADS genes encode TFs that act in a combinatorial manner in an intricate protein-protein interaction network, forming both dimeric and tetrameric complexes and driving different developmental programs. During evolution, plants have progressively diversified the MADS TF family, which likely played a role in allowing for increased plant complexity. The SEPALLATA genes appear later in evolution than other MADS floral organ identity genes and have been postulated to be key players in the evolution of flowering plants from their non-flowering ancestors ( 9). The SEPALLATA proteins form a central organising hub for MADS complex formation, particularly MADS TFs involved in flowering and floral organ development ( 10). Based on extensive genetic and biochemical studies, SEP3 is both the most promiscuous and active member of the SEPALLATA clade, able to interact with all floral organ identity MADS TFs and, when ectopically expressed with the appropriate A, B or C genes, able to convert leaves into floral organs ( 6, 11-13).

MADS TFs bind DNA as dimers through the MADS DNA-binding domain, a domain that is conserved across all eukaryotes. The addition of an alpha-helical keratin-like or 'K' domain allows plant MADS TFs to tetramerize, with the structure of the K domain of SEP3 recently characterized and the tetramerization determinants mapped at the amino acid level ( 14). A central question is whether tetramerization is required for floral organ development. One well-accepted hypothesis is that binding of tetrameric MADS TF complexes results in DNA-looping and that this change in the three-dimensional chromatin structure may be necessary for triggering expression of specific genes and therefore floral organogenesis ( 15, 16). In order to determine the role of tetramerization in flower development independently of DNA-binding, we exploited a naturally occurring splice variant of SEP3, SEP3 Δtet, which lacks a portion of the K domain (amino acids 161-174) shown to be important for homotetramerization in vitro ( 14, 17). Here, we demonstrate that SEP3 Δtet exhibits abrogated heterotetramerization capacity with different partner MADS TFs in vitro. However, the heterodimeric complexes are still able to form and are fully competent to bind DNA. Expression of SEP3 and in the sep1/2/3 mutant background under the native promoter demonstrates that while SEP3 is able to fully complement the floral phenotype, is unable to restore WT flowers. While the first three whorls (sepals, petals and stamens) harbor normal floral organs, the fourth whorl exhibits unfused carpels and an indeterminate flower-within-a-flower phenotype. The combined in vitro and in vivo experiments decouple the roles of DNA-binding from tetramer formation and demonstrate the absolute requirement of SEP3/AG tetramerization in activating genes required for floral meristem determinacy. In addition, these results suggest that tetramerization may be less crucial for floral organogenesis.

MATERIALS AND METHODS

Plant material and growth conditions

All experiments were performed using Arabidopsis thaliana in the Col-0 background. Seedlings mutated in the SEPALLATA genes ( 3), were grown in controlled growth chambers in long day conditions (16 h light/8 h dark) at 22°C for plant transformation. For in situ hybridization, western blots and qPCR analysis, plants were first grown 5 weeks in short days (8 h light/16 h dark) at 22°C, then transferred and grown for 2 weeks in long day conditions.

Plasmid construct for complementation analysis

A 6419 bp and a 6377 bp DNA fragment containing specific sequence encoding SEP3 WT (Atg24260.2) and SEP3 Δtet (Atg24260.3) respectively, under the control of SEP3 native promoter were cloned into the BamHI site of the plant expression vector pFP100 which contains the GFP gene under control of a seed specific promoter for selection ( 18). Each construct, pSEP3::SEP3 (ABRC stock number CD3-2708) and pSEP3::SEP3Δtet (ABRC stock number CD3-2709) contained 4136 bp of the SEP3 promoter region, the first exon and first intron of SEP3, exons 2-8 of the cDNA sequence for the pSEP3::SEP3 construct or exons 2-3-4-5-7-8 for pSEP3::SEP3Δtet construct, and 740 bp of the SEP3 terminator. DNA templates and primers used to amplify the constructs are listed in Supplementary Table S2.

Plant transformation and floral phenotype analysis

Sep1/2/3 triple mutants do not produce seeds. Because mutations in SEP2 and SEP3 were generated by transposon insertions ( 3), complementation analysis was performed as follows to confirm stability of the transposon mutants. The progeny of two heterozygous sep1/2/3+/− seedlings (ABRC stock number CS69927) genotyped as described ( 3, 7) and showing the expected segregation patterns with 25% of the plants exhibiting the triple mutant phenotype, were transformed by classical floral dipping methods using Agrobacterium tumefaciens-mediated gene transfer. Half of the plants were transformed with the p SEP3::SEP3 construct and the other half with the p SEP3::SEP3Δtet construct. Transformants were selected based on the fluorescence of GFP-positive seeds. More than 40 seeds carrying each transgene were put on soil and further genotyped to identify a minimum of 10 sep1/2/3 plants carrying the transgene. Primers (5′ TCAATAGGCAAGTGAC 3′ and 5′ CACTCTCTGAAGGTAGCTGAAG 3′) were used to discriminate between sep1/2/3 and sep1/2/3+/− plants carrying the construct. In parallel, untransformed control sep1/2/3 plants were genotyped from the same batch of seeds after sowing non-fluorescent (untransformed) seeds to validate the stability of the sep1/2/3 phenotype in the second generation of seeds. Floral phenotypic analyses were performed by light microscopy on flower numbers 10-19 based on their order of emergence on T1 plants genotyped sep1/2/3 for each construct and on control untransformed sep1/2/3 plants. Seeds of T1 lines expressing SEP3 or were deposited to the ABRC (stock number CS69928-31).

Protein extraction and western blot analysis

Proteins were extracted from Arabidopsis thaliana inflorescence meristems with small closed buds up to stage 10-11 ( 19). Between 20 and 40 mg of tissues were ground in liquid nitrogen and resuspended in 2X Coomassie loading buffer (50 μl per 100 mg tissue, diluted in 100 mM Tris buffer, pH 7.5, supplemented with an antiprotease cocktail (Roche)). After 5 min at 95°C, samples were centrifuged 5 min, and 8 μl of total soluble proteins were separated on a SDS 15% polyacrylamide gel and transferred to a Hybond LFP PVDF transfer membrane (GE Healthcare). Western blot analyses were performed using the SEP3 antibody ( 20) at a 1:1000 dilution. Detection was performed using the Clarity Western ECL Substrate kit (BIO-RAD). Equal loading of the samples was checked by Coomassie blue protein detection on a separate gel.

Environmental scanning electron microscopy

SEM experiments were performed at the Electron Microscopy facility of the ICMG Nanobio-Chemistry Platform (Grenoble, France). Untreated flowers were directly placed in the microscope chamber. Care was taken to maintain some humidity during the pressure decrease in the chamber in order to prevent tissue drying. Secondary electron images were recorded with a Quanta FEG 250 (FEI) microscope while maintaining the tissue at 2°C, under a pressure of 500 Pa and a 70% relative humidity. The accelerating voltage was 14 kV and the image magnification ranged from 100 to 800x. Flowers from 3 independent lines were observed for each genotype.

Gene expression analysis by real time PCR

RNA was isolated from Arabidopsis thaliana Col-0 inflorescence meristems with small closed buds up to stage 10-11 ( 19) using RNAeasy plant mini kit (Qiagen). Complementary DNA (cDNA) was primed with random hexamers on 1μg DNase-treated RNA using Superscript III Reverse Transcriptase (Thermo Fisher Scientific). Real time qRT-PCR was performed on a CFX Connect real-time system (BIO-RAD) using Sso Advanced Universal SYBR Green supermix (BIO-RAD) and specific primers listed in Supplementary Table S2. Three technical replicates were run in parallel per sample. Quantification of gene expression was performed using the comparative CT method with the CFX Connect Manager 3.1 software. The ACTIN2 (At3g18780) and the EF-1α (At1g09740) genes were used to normalize the qPCR data ( 21). Statistical analyses were performed by Student's t-test.

In situ hybridization

RNA in situ hybridization was performed as previously described ( 22). Briefly, inflorescences were harvested 2 weeks after bolting. DIG-labeled AG antisense probe was generated by T7 RNA polymerase activity from a 1-kb insert cloned into the pBS KS+ vector and digoxigenin-labeling mix (Roche).

EMSA experiments

AG (At4g18960.1), SEP3 WT (At1g24260.2) and SEP3 Δtet (At1g24260.3) cDNAs were PCR amplified using specific primers ( Supplementary Table S2), cloned into the pSP64 plasmid (Promega) and used for in vitro transcription translation (Promega SP6 High Yield Expression System). AP3 (At3g54340) and PI (At5g20240) were cloned into pSPUTK ( 23). EMSAs were performed as described ( 17) with 10 nM DNA labeled with Cy5 (Eurofins). Primers used to amplify DNA probes and probe sequences are listed in Supplementary Tables S2 and S3. For each EMSA, a negative control was run, labeled 'DNA alone' in which the in vitro translation assay was done with pSP64 vector without any insert and incubated with the DNA probe.

Yeast two-hybrid

The cDNAs encoding SEP3, SEP3Δtet, AP3, PI and AG were cloned into both pGBKT7 and pGADT7 vectors (Clontech), using the NdeI/PstI and NdeI/XhoI restriction sites, respectively. The bait and prey vectors were transformed into yeast strain Y187 and AH109, respectively. After mating, the resulting diploids were selected with medium lacking Leu and Trp (-LW). The selected strains were dotted at two different optical densities (OD ₆₀₀ of 2 and 20) onto two different selection plates containing -LW or -LWAH medium (lacking Leu, Trp, Ade and His), the latter used to select diploids presenting positive protein interaction. Screening was done in duplicate.

Homology modeling

Sequence alignments and secondary structure prediction of SEP3 and AG were performed with Clustal and JPred4, respectively, followed by homology modeling of the SEP3/AG heterotetramer using the structure of the SEP3 homotetramerization domain (4OX0) and threading with the AG sequence ( 24-26). The models were inspected in Coot and side chain conformations manually optimized for hydrogen bonding ( 27). All structural figures were generated with PyMOL ( 28).

Bioinformatics analysis

ChIP-seq data for SEP3 ( 29) and AG ( 30) were downloaded from Arrayexpress and mapped and normalized using bowtie1.2.1 ( 31) and MACS2.1.1 ( 32). MNase data ( 33) were mapped using bowtie1.2.1. For Segal scores ( 34), 10 000 bp were added to the flanking region of each gene and computed using the web server ( https://genie.weizmann.ac.il/software/nucleo_prediction.html). Predicted binding sites for SEP3 were computed using the position weight matrices from JASPAR ( 35) and the genes scanned using FIMO from the MEME suite ( 36) with P-value < 10e-4.

RESULTS

SEP3 and SEP3Δtet bind DNA in vitro

SEP3 Δtet lacks residues 161-174 in the K domain, breaking the alpha helix that forms the major interaction surface necessary for stable tetramerization (Figure 1A and B). Using in vitro transcription-translation, the DNA-binding and oligomerization capabilities of the SEP3 and SEP3 Δtet proteins were determined via electrophoretic mobility shift assays (EMSAs) ( 14, 23, 37). Similar production of SEP3 and SEP3 Δtet proteins using the in vitro transcription-translation assay was confirmed by western blot analysis ( Supplementary Figure S1A). As shown in Figure 1C, using a DNA fragment from the SEP3 promoter with two SEP3 binding sites (CArG boxes), the protein can bind DNA on a single site as a dimer or as a higher order complex with four molecules of SEP3 bound per DNA, suggesting cooperativity between the two dimers due to homotetramerization of the protein. This higher order complex was absent when only one CArG box was present ( Supplementary Figure S1B). In contrast, the ability of SEP3 Δtet to form this higher order complex is virtually abolished, demonstrating dramatically reduced cooperative binding by SEP3 Δtet (Figure 1C). These data indicate that SEP3 Δtet can efficiently bind DNA as a homodimer and recognizes the CArG box motif. However, cooperativity and tetramer formation is strongly impaired. SEP3 Δtet was then used as a tool to study the impact of tetramerization on flower development in vivo.

restores petals and stamen identity in the sep1/2/3 triple mutant background but not meristem determinacy

As the single and double sepallata mutants do not have strong phenotypes, the sep1/2/3 triple mutant background was chosen to investigate function. The sep1/2/3 mutant exhibited conversion of all floral organs to sepaloid-like structures and a lack of floral meristem determinacy ( 3). Arabidopsis Col-0 plants were transformed with either SEP3 or under the control of the native promoter to generate sep1/2/3 pSEP3::SEP3 (hereafter referred to as sep1/2/3 SEP3) and sep1/2/3 pSEP3:: SEP3Δtet (hereafter referred to as sep1/2/3 SEP3Δtet) (Figure 2). Phenotypic complementation, defined by flowers having sepals, petals, stamens and carpels in whorls 1-4, respectively, with normal siliques harbouring seeds, was found in the overwhelming majority of T1 sep1/2/3 plants (15 of 18) expressing wild type SEP3 (Figure 2C, G, K). The remaining three T1 plants exhibited the classic sep1/2/3 phenotype. Conversely, 9 of 11 T1 sep1/2/3 plants expressing the construct showed incomplete flower development. While petals and stamens were clearly observed in these plants, two unfused carpeloid-like structures were produced instead of the WT gynoecium in whorl 4 (Figure 2D, H, L, M, O). Further inspection revealed that instead of containing ovules and seeds, whorl 4 exhibited supernumerary floral organs characteristic of the flower within-a-flower phenotype (Figure 2N, P). An extended gynophore was observed in these plants and all plants were sterile. The two remaining T1 plants carrying the vector exhibited the sep1/2/3 mutant phenotype. Petal number in both lines expressing either SEP3 or was identical to WT, while the number of stamens was slightly reduced for both lines compared to WT (5.8 ± 0.4 for WT versus 4.5 ± 0.2 for sep1/2/3 SEP3 lines and 4.5 ± 0.4 for sep1/2/3 SEP3Δtet lines). Among 18 plants genotyped sep1/2/3 in the pool of the untransformed T1 plants, 16 showed the characteristic triple mutant phenotype (Figure 2B, F and J) and none exhibited the sep1/2/3 SEP3Δtet phenotype. The remaining two plants showed the WT phenotype. These phenotypes were recapitulated in the T2 generation for the SEP3 and lines. In the case of plants expressing plants were selected from the T1 and sep1/2/3 plants carrying the transgene were selected in the T2 generation. The T2 generation was used for all subsequent experiments.

In order to determine if the observed phenotypes were due to differences in protein levels, western blots using a SEP3-specific antibody was performed on independent lines for each construct. SEP3 and SEP3 Δtet accumulate at similar levels to WT SEP3 in all transformed lines ( Supplementary Figure S2). These data suggest that the differences in whorl 4 between sep1/2/3 expressing SEP3 or were unlikely to be due to protein accumulation level, but rather due to bona fide differences in protein function.

restores carpel cells in the sep1/2/3 mutant background but not the production of nectaries

To better characterize the floral organ identity in the triple mutant plants expressing , scanning electron microscopy experiments were performed. The cell surface morphology of all the floral organs in the sep1/2/3 mutant has a sepaloid-like character (Figure 3 and Supplementary Figure S3) as previously described ( 4). In contrast, the distinct cell surface morphology of sepals, petals and stamens were observed in WT and sep1/2/3 plants expressing SEP3 or ( Supplementary Figure S3). Inspection of the cell surface in the fourth whorl in these lines reveals, similar to WT, stigmatic papillae, valve cells, style and replum (Figure 3A, B, E). This indicates that is able to launch carpel development. However, the gynoecium is unfused (Figure 3E). The interior of the carpel structure reveals a cell surface morphology characteristic of stamen and carpels in sep1/2/3 SEP3Δtet plants (Figure 3G). In addition, no nectaries were observed in plants (Figure 3L) but were present in the WT (Figure 3I) and SEP3 complemented lines (Figure 3K). These data show that the expression of fully restores petal and stamen development. In contrast, while the carpel development program was launched, gynoecium development is incomplete due to a lack of determinacy of the floral meristem. We questioned whether the indeterminate floral phenotype observed could be due to a downregulation of AG as this gene is a known regulator of floral meristem determinacy through the repression of the stem cell maintenance gene WUSCHEL (WUS) ( 38-40).

does not impact AG expression but misregulates KNU and CRC

SEP3 binds to the promoter of AG, suggesting the possibility that the lack of determinacy due to could be due to downregulation or misregulation of AG. As shown in Figure 4A, AG transcript levels in IMs and closed flower buds in WT, mutant and transformed lines were indistinguishable. In situ hybridization also confirmed that the early pattern of AG expression, in the centre of the floral meristem and in the primordia of whorls three and four, was similar in all lines (Figure 4B). This is consistent with previous reports for the sep1/2/3 mutant that also reported unaltered early AG expression with respect to WT ( 3). Thus, does not have a dramatic impact on AG expression levels and seems unlikely to account for the observed indeterminacy phenotype. Additional qRT-PCR experiments were performed on the floral MADS genes AP3, PI, AP1 and SEP4 to determine if there was misregulation of other floral organ identity genes, however no unexpected differences were observed ( Supplementary Figure S4).

Since AG expression was not significantly altered, we examined downstream targets of AG as possible candidates for the observed indeterminacy phenotype. AG has been previously shown to activate KNUCKLES (KNU) and CRABSCLAW (CRC) ( 40-46), known repressors of WUS. As shown in Figure 4C, KNU expression was significantly lower in sep1/2/3 compared to WT, and in sep1/2/3 expressing compared to either the double sep1/2 mutant or the triple mutant expressing SEP3. Interestingly, sep1/2 mutant and transformed lines showed higher expression levels of KNU compared to WT, suggesting a misregulation of KNU. KNU has significant expression in stamen and ovules in addition to its expression in the floral meristem ( 47). Different SEPALLATA proteins may play slightly different roles in regulating KNU in specific floral tissues, resulting in the observed KNU expression levels in sep1/2 mutants. CRC is required for nectary formation and, as expected by the absence of nectaries in sep1/2/3 and plants, CRC expression was strongly decreased in these lines and restored to WT and sep1/2 levels when the sep1/2/3 triple mutant was complemented with SEP3 but not when transformed with (Figure 4D). These experiments demonstrate that both KNU and CRC are specifically misregulated in the triple mutant expressing , with CRC showing the clearest downregulation.

Interaction patterns of SEP3Δtet and MADS floral organ identity TFs

To better understand the ability of SEP3 Δtet to restore floral organ formation but not determinacy, we first compared the ability of SEP3 and SEP3 Δtet to interact with AG by yeast-2-hybrid. Interactions for SEP3 and SEP3 Δtet with AG were positive, indicating that SEP3 Δtet can interact with AG in vivo ( Supplementary Figure S5). The binary interactions of SEP3, AP3 and PI were also tested and were consistent with published reports ( 10) ( Supplementary Table S1). The interaction of SEP3 Δtet and AG was also validated by EMSA, using a single CArG box showing that heterodimers formed and bound DNA ( Supplementary Figure S6).

To further decipher the interaction patterns and oligomerization state of SEP3 and SEP3 Δtet, EMSAs were performed with SEP3 or SEP3 Δtet and the different floral organ identity interaction partners AG, AP3 and PI using a DNA fragment with two binding sites. As shown in Figure 5A, the tetrameric interaction of SEP3 Δtet with AG was strongly impaired. At the lowest protein concentration used, SEP3 Δtet/AG complexes were predominantly dimeric, resulting in the disappearance of the higher molecular weight band observed for SEP3/AG. Titrating increasing protein concentrations gave rise to a slight amount of higher molecular weight complex for SEP3 Δtet/AG, however, the predominant species was a dimer under all conditions tested, indicating lack of cooperative binding by SEP3 Δtet/AG ( Supplementary Figure S7). In the presence of AP3 and PI, while SEP3/AG tetramers are favored (Figure 5B, lanes 3 and 5), the putative tetrameric complexes with SEP3/AP3/PI/AG were formed (Figure 5B, lane 7). In the presence of SEP3 Δtet, SEP3 Δtet/AG heterodimers predominate (Figure 5B, lane 4) with a decreased amount of SEP3 Δtet/AG/AP3/PI complex formed as compared to SEP3/AG/AP3/PI, indicating reduced cooperative binding and impaired tetramerization. In the absence of SEP3 or SEP3 Δtet, an AG/AP3/PI complex of higher molecular weight was observed, suggesting full occupancy of the available MADS binding sites and possible formation of a higher order complex even in the absence of SEP3. Overall, SEP3 Δtet was strongly impaired in its ability to bind cooperatively and form tetrameric complexes with its MADS partners. As interactions with AG were most affected by the SEP3 Δtet mutation and the observed developmental defects were all in the fourth whorl, the interaction patterns of SEP3 Δtet/AG were further explored in the context of the KNU and CRC promoters.

In vitro binding of SEP/AG to the KNU and CRC promoters

The promoters of both KNU and CRC contain multiple putative binding sites for AG and SEP3 based on ChIP-seq data and binding site prediction ( http://jaspar.genereg.net/) ( 29, 35, 42, 48) (Figure 6). Furthermore, extensive characterization of the KNU promoter has identified binding sites for AG that overlap with Polycomb response elements (PRE) ( 49) and with predicted SEP3 binding sites. As shown in Figure 6A and B, the AG and SEP3 ChIP-seq binding sites overlap in the KNU and CRC promoters, respectively. Based on these data, promoter sequences from KNU and CRC, each with two putative binding sites for SEP3 and AG, were used for EMSA experiments and the binding patterns shown in Figure 6C. For both the KNU and CRC promoters, SEP3/AG showed robust cooperativity and tetramerization, with the majority of the bound species exhibiting a higher molecular weight shift as compared to the dimeric species. In contrast, SEP3 Δtet/AG exhibited dramatically reduced cooperativity and higher order complex formation, with the major species corresponding to a single SEP3 Δtet/AG dimer. These results indicate that impaired tetramerization of SEP3 Δtet with AG affects the in vitro binding of the proteins at the promoters of KNU and CRC and provides a molecular basis for the observed phenotypes for SEP3 and in the triple mutant background.

DISCUSSION

The quartet model of floral organ formation presupposes the formation of tetrameric MADS complexes as critical to the proper regulation of downstream target genes, putatively through DNA looping ( 10, 15, 16). Here, we begin to decouple the role of DNA-binding from tetramerization by investigating the activity of a naturally occurring splice variant, SEP3 Δtet, which we predicted would be impaired in tetramerization based on structural and biochemical studies ( 14, 17). While the physiological role of SEP3 Δtet is not known, SEP3 undergoes temperature dependent alternative splicing, with the variant showing increased expression at lower temperatures ( 50). Other MADS genes also exhibit temperature dependent alternative splicing, including the flowering time genes FLOWERING LOCUS M ( FLM) and MADS AFFECTING FLOWERING2 ( MAF2) ( 37, 51, 52). The FLM and MAF2 isoforms expressed preferentially at lower ambient temperatures are able to form complexes with the MADS TF, SHORT VEGETATIVE PHASE (SVP), and delay flowering via repression of the downstream floral pathway integrators, SUPRESSOR OF CONSTANS OVEREXPRESSION 1 (SOC1) and FLOWERING LOCUS T (FT). However, increased ambient temperature results in FLM and MAF2 isoforms that are unable to repress FT and SOC1. Indeed, temperature dependent alternative splicing may be a mechanism used more generally by MADS TFs to tune their activity in response to temperature. One could speculate that the SEP3 Δtet isoform may have a role in titrating SEP3 activity in a temperature dependent manner by favoring dimeric versus tetrameric complex formation. Our previous overexpression studies with in the WT background demonstrated early flowering and a floral phenotype characterized by perturbations in floral organ number ( 50), however interpreting the role of the SEP3 Δtet isoform in the WT background was not possible due to the presence of the four SEP proteins and the role of tetramerization could not be fully addressed. In order to more fully explore the role of SEP3 tetramerization on flower development and differentiate the function of SEP3-containing dimeric versus tetrameric MADS complexes, transformation of in the sep1/2/3 background was required.

Surprisingly, sep1/2/3 plants expressing SEP3 or demonstrate a requirement for robust SEP3-driven tetramerization of MADS TF complexes in floral meristem determinacy but seemingly not petal, stamen and carpel identity. SEP3 Δtet is able to fulfil the majority of regulatory functions of the SEP3 isoform, including activation of genes required for floral organ specification ( 4, 6). However, SEP3 Δtet did not complement the indeterminacy phenotype. Indeed, the continued proliferation of meristematic cells may point to a physiological function of the SEP3 Δtet isoform in delaying premature floral meristem termination under low temperature conditions in which cell division is slower, however this remains to be determined. While the putative physiological role of the SEP3 Δtet isoform is still speculative, sep1/2/3 plants expressing demonstrate a clear and reproducible phenotype with complementation of floral organ identity and defects limited to an indeterminacy phenotype. Complementation of floral organ identity defects may be due to the residual tetramerization capabilities of different combinations of MADS TF partners. Indeed, the floral organ identity tetramers for petal and stamen development putatively contain only a single SEP3 or SEP3 Δtet and the presence of three other MADS partner proteins may exercise a compensatory effect on tetramerization and cooperative DNA-binding. We observed that, for the same DNA sequence, the formation of a higher molecular weight species corresponding to four MADS TFs (SEP3 Δtet / AP3/PI/AG) was less affected than with two MADS TFs (SEP3 Δtet /AG), (Figure 5). In addition, while the in vitro studies described suggest that tetramerization is impaired with SEP3 Δtet/AG/AP3/PI, in vivo conditions may favor or stabilize these weak tetrameric MADS complexes or ternary factors may contribute to higher order complex formation. Conversely, tetramer formation may not be absolutely required for activation of target genes during floral organ development with simply the correct dimeric species formed between the A, B, C and E partners and occupancy of the appropriate CArG box binding sites. This second hypothesis is likely in the case of carpel organogenesis as tetramer formation with AG was strongly impaired with SEP3 Δtet and cooperative DNA binding dramatically affected, yet the fourth whorl cells adopted carpel characteristics including clear development of the stigma, valve and replum. To fully understand whether dimeric species are sufficient to trigger floral organ development further mutational studies in vitro and in vivo of multiple MADS TFs will need to be performed with complete abrogation of tetramerization and cooperative DNA binding for all partner combinations.

Unfused carpel and extended gynophore together with lack of determinacy are phenotypes observed in sep1/2/3 expressing and described for crc and knu mutants, respectively ( 47, 53). CRC and KNU are known to belong to two parallel pathways regulating carpel development and determinacy, are directly activated by AG and also have SEP3 binding sites in their promoter regions ( 40, 41, 49, 54). Gene expression of CRC and KNU is downregulated in the sep1/2/3 triple versus the sep1/2 double mutant and is restored back to sep1/2 levels of expression in sep1/2/3 SEP3 lines, but not in sep1/2/3 SEP3Δtet lines. This shows a trend with the tetramerization competent SEP3 able to more strongly activate KNU and CRC versus SEP3 Δtet. One cannot rule out the possibility that the 14-amino acid deletion in SEP3 Δtet results in the loss of a ternary factor interface that impedes recruitment of additional proteins needed for activation of floral meristem determinacy genes. However, consistent with our results indicating SEP3/AG tetramerization is crucial for floral meristem determinacy ( 55, 56), the examination of different ag mutant alleles, AG-Met205 and ag-4, weak and strong mutant alleles of AG, respectively, exhibit similar phenotypes to sep1/2/3 SEP3Δtet. AG-Met205 has meristem determinacy defects but no defects in stamen organogenesis or carpel specification ( 55). The ag-4 allele shows a stronger phenotype with stamen in the third whorl and indeterminacy in the fourth whorl with no carpel formation. Analysis of these mutant alleles reveals that AG-Met205 has a lysine to methionine mutation that breaks a putative salt bridge important for tetramerization with SEP3 ( Supplementary Figure S8). For ag-4, a portion of the K domain is deleted due to skipping of exon 6, resulting in loss of the tetramerization interface while preserving dimerization and DNA-binding ( Supplementary Figure S8). These data demonstrating that both AG and SEP3 Δtet tetramerization interface mutants share similar phenotypes provides additional evidence that the SEP3/AG tetramer is absolutely required for floral meristem determinacy.

A fundamental question remains as to why SEP3/AG tetramers are required for KNU and CRC activation and seem to play a lesser role in floral organogenesis. One possible explanation is the strong repression of both these genes prior to flower development (Figure 6A and B). AG activation of KNU requires the eviction of the Polycomb repressive complex 2 (PRC2) from the KNU promoter. AG binding sites overlap with putative Polycomb response elements (PRE), and direct competition for these binding sites between AG and PRC2 is thought to play a role in KNU activation by AG ( 49). If AG must outcompete PRC2 for binding to the KNU promoter, robust tetrameric interactions with SEP3 may be required. Likewise, the CRC promoter shows strong nucleosome positioning based on MNase experiments ( 33) and is bound by the PRC2 interacting protein EARLY EMBRYONIC FLOWER1 (EMF1) in seedlings, indicating that this gene is in a closed region of chromatin ( 57). After SEP3 binding, this closed region was shown to open, triggering CRC expression ( 29). Thus, the inability of SEP3 Δtet to fully restore CRC and KNU expression in the triple mutant background suggests that tetrameric SEP/AG complexes are needed to overcome strong repression. This may be due to an important cooperativity or avidity effect, with impaired tetramerization resulting in decreased overall occupancy of the SEP3 Δtet/AG complex at its cognate sites. A second intriguing possibility for the misregulation of KNU and CRC by the SEP3 Δtet /AG complex is the lack of DNA-looping ( 15, 16, 23). The abrogation of tetramerization eliminates SEP3 driven DNA-looping. DNA-looping is known to be important in gene regulation ( 58), although it is still controversial the extent of DNA-looping engendered by the MADS TF as few chromatin conformation capture studies with sufficient resolution have been performed in plants ( 59, 60). Identification of transient short-range DNA loops by tetrameric MADS complexes will require further investigation to fully address this question.

These in vitro and in vivo experiments provide novel data as to the requirements of tetramerization for MADS TF function in flower development. The absolute requirement for strong tetrameric interactions may be limited to robustly repressed targets (such as CRC and KNU) as floral organ identity programs were successfully launched in the sep1/2/3 triple mutant expressing , including carpel development in the fourth whorl. The combination of experiments presented here refines the model for flower and floral organ development and sheds new light on the role of the SEPALLATA MADS TFs in triggering different developmental programs.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

ACKNOWLEDGEMENTS

The authors would like to thank K. Kaufmann and C. Smaczniak for plasmids, SEP3 antibody, helpful discussions and advice, B. Davies, M. Yanofsky and J. Ripoll for sep mutant seeds, and G. Tichtinsky, G. Vachon and R. Dumas for helpful discussions.

FUNDING

Action Thématique et Incitative sur Programme (ATIP)-Avenir (to C.Z.); Grenoble Alliance for Integrated Structural Cell Biology (ANR-10-LABX-49-01 to V.H., A.J., C.Z., Q.C., A.S., V.C., F.P.); Agence Nationale de la Recherche (project Flopinet to C.Z., V.H., A.J., F.P., C.C.) and Centre National de la Recherche Scientifique (to S.J.C.). Funding for open access charge: ATIP-Avenir, ANR.

Conflict of interest statement. None declared.

REFERENCES

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# data[].attributes.attachment_link.title
Tetramerization of MADS family transcription factors SEPALLATA3 and AGAMOUS is required for floral meristem determinacy in Arabidopsis | Nucleic Acids Research | Oxford Academic

# data[].attributes.attachment_link.description
The MADS transcription factors (TF) constitute an ancient family of TF found in all eukaryotes that bind DNA as obligate dimers. Plants have dramatically expanded the functional diversity of the MADS family during evolution by adding protein-protein interaction domains to the core DNA-binding domain, allowing the formation of heterotetrameric complexes.