In planta localisation patterns of MADS domain proteins during floral development in Arabidopsis thaliana
© Urbanus et al; licensee BioMed Central Ltd. 2009
Received: 01 October 2008
Accepted: 12 January 2009
Published: 12 January 2009
MADS domain transcription factors play important roles in various developmental processes in flowering plants. Members of this family play a prominent role in the transition to flowering and the specification of floral organ identity. Several studies reported mRNA expression patterns of the genes encoding these MADS domain proteins, however, these studies do not provide the necessary information on the temporal and spatial localisation of the proteins. We have made GREEN FLUORESCENT PROTEIN (GFP) translational fusions with the four MADS domain proteins SEPALLATA3, AGAMOUS, FRUITFULL and APETALA1 from the model plant Arabidopsis thaliana and analysed the protein localisation patterns in living plant tissues by confocal laser scanning microscopy (CLSM).
We unravelled the protein localisation patterns of the four MADS domain proteins at a cellular and subcellular level in inflorescence and floral meristems, during development of the early flower bud stages, and during further differentiation of the floral organs. The protein localisation patterns revealed a few deviations from known mRNA expression patterns, suggesting a non-cell autonomous action of these factors or alternative control mechanisms. In addition, we observed a change in the subcellular localisation of SEPALLATA3 from a predominantly nuclear localisation to a more cytoplasmic localisation, occurring specifically during petal and stamen development. Furthermore, we show that the down-regulation of the homeodomain transcription factor WUSCHEL in ovular tissues is preceded by the occurrence of both AGAMOUS and SEPALLATA3 proteins, supporting the hypothesis that both proteins together suppress WUSCHEL expression in the ovule.
This approach provides a highly detailed in situ map of MADS domain protein presence during early and later stages of floral development. The subcellular localisation of the transcription factors in the cytoplasm, as observed at certain stages during development, points to mechanisms other than transcriptional control. Together this information is essential to understand the role of these proteins in the regulatory processes that drive floral development and leads to new hypotheses.
Major developmental steps in flowering plants, such as the transition to flowering and floral organ development are, for the most part, controlled by members of the MADS domain family of transcription factors . The action of these transcription factors in defining the identity of the floral organs has been captured in a genetic model, the "ABC" model , which was later extended with a "D" and "E" function [3–6]. This model describes how the combinatorial activity of several classes of regulatory genes, most of which encode MIKC-type MADS domain proteins, define the identity of the five different floral organs (sepals, petals, stamen, carpels, and ovules). According to this model, the combination of the class A+E genes specifies the identity of sepals, while the A+B+E genes specify petal identity, the combination of classes B+C+E determines stamen identity, C+E genes together lead to carpel identity, and finally the combination of classes C+D+E is responsible for ovule identity (for review see ). Floral organ development in Arabidopsis thaliana is controlled by the following genes: the A-function is represented by the genes APETALA1 (AP1) and APETALA2 (AP2) (not a MADS domain transcription factor); the B-function is controlled by APETALA3 (AP3) and PISTILLATA (PI); AGAMOUS (AG) represents the C-function; the D-function is represented in a redundant manner by SEEDSTICK (STK), SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2); and the E-function involves the four closely related genes SEPALLATA1 to SEPALLATA4 (SEP1-4). Furthermore, another MADS box gene FRUITFULL (FUL), which is not described in the "ABC" model, is also involved in carpel development. In addition to these functions in floral organ development, some of these genes also have other functions. For instance, both FUL and AP1 are involved in the transition from inflorescence meristem to floral meristem identity, while AG controls the floral meristem determinacy [8–10].
The MADS domain proteins and the "ABC" model are well studied subjects for transcription factor regulation and action. Several studies have shown that at least some MADS domain proteins need to be in a dimeric form, either homo- or heterodimeric, before they can enter the nucleus [11–13]. In the nucleus, the proteins bind to DNA sequences of the target gene with the consensus CC(AT)6GG sequence, also known as the CArG box . Binding to the DNA occurs either in the form of a dimer  or in a multimeric fashion [15, 16], for instance in a tetrameric form as proposed in the "quartet" model . Our knowledge about these MADS domain protein interactions has been greatly extended by a study where interaction data obtained from a systematic Yeast Two-Hybrid experiment was combined with large scale microarray co-expression data of the corresponding genes . By considering not only the capacity of proteins to interact with each other, but also the possibility for putative partners to be co-localised in the same tissues and cells, the output is narrowed down to interactions that are likely to be of biological relevance for the plant.
However, microarray studies give only a very broad view on the spatio-temporal expression pattern of genes and do not provide the necessary detail that is needed to demonstrate co-localisation of the encoded proteins. In situ mRNA hybridisation studies and promoter-reporter studies like the ones reported for AG , SEP3 , AP1 , and FUL  reveal the expression patterns in more detail, but these might not reflect the protein localisation patterns. In fact, it is difficult to infer the protein localisation pattern from an mRNA expression pattern for two main reasons. First of all, production and degradation rates of mRNA and proteins could be totally different, and secondly, proteins can be transported from an expressing cell to a neighbouring non-expressing cell; all resulting in protein patterns that deviate from the mRNA patterns. It is known from studies on the class B genes DEFICIENS and GLOBOSA from Antirrhinum majus  that particular MADS domain transcription factors are able to transfer from one cell to another, where they may have a non-cell-autonomous function. Therefore, to obtain information about the spatio-temporal control of these regulatory proteins, it is essential to study the localisation of the proteins themselves. Additionally, protein localisation studies can be more informative on the functioning of transcription factors by showing the specific subcellular localisation of the proteins during development. It has been demonstrated that some MADS domain transcription factors are localised in the cytoplasm when an interaction partner is absent and only become functional when they enter the nucleus after dimerisation [11–13, 24]. Preferably, one would like to obtain a three-dimensional map of protein localisations with cellular resolution and information about the dynamics of proteins during plant development. The discovery of GREEN FLUORESCENT PROTEIN (GFP) and similar fluorescent proteins and their use as visual tags for proteins, in combination with Confocal Laser Scanning Microscopy (CLSM) has made this visualisation of fluorescently tagged proteins in living plant tissue possible [25–27].
In order to study MADS domain proteins in living tissues with CLSM during floral development, we made C-terminal GFP tagged versions of SEP3, AG, FUL, and AP1. Previously it was shown that the fusion of GFP to the C-terminus of the MADS domain protein AP1 does not affect its function, as it is able to complement the ap1 mutant . Furthermore, it is known from studies with AG, STK, and SEP3 that introns can contain important regulatory elements that are required for the correct expression pattern of these MADS box genes [29–31]. For this reason, we made C-terminal GFP tagged versions of SEP3, AG, FUL, and AP1 using genomic fragments . Here we describe the protein localisation patterns of gSEP3:GFP, gAG:GFP, gFUL:GFP and gAP1:GFP on a cellular and subcellular level in the inflorescence meristem and at various stages of floral meristem and organ formation. This detailed study reveals discrepancies between the previously reported mRNA expression patterns and the protein localisations, and sheds new light on the functioning of the MADS domain proteins in floral organ patterning and formation.
Results and discussion
In this study we analysed the spatio-temporal protein localisation patterns of C-terminally GFP tagged genomic clones of MADS domain proteins SEP3, AG, FUL and AP1 during floral development, hereafter referred to as SEP3:GFP, AG:GFP, FUL:GFP, and AP1:GFP. The generated constructs were transformed to Arabidopsis thaliana wild type Col-0 plants, and at least four GFP-expressing stable primary transformants per construct were analysed for their protein localisation. These were found to be very similar in localisation patterns, although some differences in expression levels were observed. These differences in expression levels may be due to differences in transgene copy numbers, but they may also be caused by positional effects of the insertion of the transgene. Three constructs, namely AG:GFP, FUL:GFP and AP1:GFP, were also introduced into their respective mutant lines. These complementation experiments showed that C-terminal GFP tagged MADS domain proteins are functional, as the AG:GFP, FUL:GFP and AP1:GFP proteins can rescue the mutant phenotypes of ag, fu1 and ap1 mutants, respectively. As the single sep3 mutant shows very subtle phenotypic alterations due to the redundancy of SEP3 with SEP1 and SEP2 , the SEP3:GFP construct was only transformed into the wild type background. The spatio-temporal protein localisation patterns described here are from representative lines homozygous for the transgene in the Col-0 wild type background.
SEP3 localisation in inflorescence meristem and early flower bud stages
Interestingly, the subcellular localisation of the SEP3:GFP protein changed dramatically from stage 3 onwards. Just before the petal and stamen initiation, SEP3:GFP proteins in the future second and third whorl became both cytoplasmically and nuclear localised. At the same time, the proteins in the innermost part of the floral meristem clearly remained nuclear localised (Figures 1E and 1F). This cytoplasmic SEP3:GFP signal in the second and third whorl could be due to higher expression levels of the gene, resulting in a temporary accumulation of SEP3:GFP proteins in the cytoplasm waiting for transportation to the nucleus. Another option could be that the appearance of new interaction partners in this region results in the cytoplasmic localisation of SEP3:GFP. For instance, it is known that the expression of AP3, which determines petal and stamen identity together with PI and SEP3, also starts at stage 3 and is restricted to the same area where whorl 2 and 3 will develop . It would be interesting to investigate if AP3 is also located predominantly in the cytoplasm at this point in development. Another possibility could be that higher cell division rates in the area of the future second and third whorl which cause the petal and stamen primordia to arise, result in increased unloading of previously nuclear localised proteins into the cytoplasm . The smaller cell sizes in the area of the future second and third whorl compared to the innermost part of the floral meristem could indicate higher cell division rates (Figure 1E). This unloading of the nuclear localised proteins into the cytoplasm during cell division may also allow the proteins to meet new partners and form new complexes in the cytoplasm, for instance with AP3 and PI. In stage 5 flower buds, the initiating petal and stamen primordia revealed an epidermal layer with mostly nuclear SEP3:GFP, while the subepidermal and inner cell layers showed a lower SEP3:GFP signal, both cytoplasmically and nuclear localised (Figures 1G–I). Apparently, SEP3 is less needed in the inner layers of the emerging petals and stamen than in the epidermis. In the mean time the SEP3:GFP proteins in the fourth whorl remained nuclear localised (Figure 1H).
AG localisation in inflorescence meristem and early flower bud stages
FUL localisation in inflorescence meristem and early flower bud stages
FUL mRNA is expressed in two distinct phases: in the inflorescence meristem and in the centre of the floral meristem from stage 3 flower bud onwards, but not in the intermediate flower bud stages 1 and 2 [22, 36]. Therefore, the FUL:GFP presence that was observed in flower bud stages 1 and 2 (Figures 3A and 3B) might be due to FUL:GFP protein that remained from previous stages. The renewed FUL gene expression in stage 3 flower buds in the centre of the floral meristem corresponds with the increased FUL:GFP protein accumulation in the centre of the floral meristem from stage 3 onwards (Figure 3C). In summary, throughout the development from inflorescence meristem to floral organs it seems that the tissues that need to remain undifferentiated, such as the inflorescence and floral meristem, have FUL:GFP protein in all cell layers. Differentiating tissues however, like the developing floral organs, seem to loose the subepidermal and inner cell layer FUL:GFP signal and only retain the signal in the epidermis.
AP1 localisation in inflorescence meristem and early flower bud stages
SEP3, FUL and AP1 localisation during petal development
The function of the MADS domain proteins in the floral transition and in the determination of floral organ identities is well established, however, less is known about putative later functions of MADS domain proteins in differentiating floral organs. Recently, a report showed that AG has a late function in stamen development , and it was already known that FUL has a late function in pistil and fruit development [22, 39]. In view of this and the fact that MADS domain proteins were still present in differentiating floral organs, we studied the SEP3:GFP, FUL:GFP and AP1:GFP localisations during the development of the petal in more detail.
FUL, SEP3 and AG localisation during pistil development
It is well-known that FUL has a late function in valve differentiation in the developing pistil and in fruit elongation [22, 39]. In contrast, much less is known about putative late functions of SEP3 and AG during pistil development. Therefore, we studied the localisation patterns of FUL:GFP, SEP3:GFP and AG:GFP in the developing pistil until flower bud stage 12 (stage after ), when style, valves, valve margins, and replum are being formed. Note that in stage 12 pistil tissue the confocal microscope laser could not reliably penetrate beyond five cell layers.
SEP3:GFP fusion protein is most abundant in the replum and the valve margins, while the valves and the style showed a much lower signal (Figures 6B and 6D). In the replum and the valve margins SEP3:GFP was located in the first two cell layers, whereas the style and the valves had only signal in the epidermal layer at this stage of pistil development (Figure 6F). However, in a younger gynoecium (stage 10) the same localisation pattern with lower intensity was found, but at least three cell layers deep in the future style and two cell layers deep in the future valve margins, the future replum, and the future valves (data not shown). Therefore, it seems that during the development of the gynoecium SEP3:GFP becomes restricted to the epidermal and subepidermal layers.
We observed that AG:GFP is present throughout all cell layers of the whole pistil including the style and stigma (Figures 6C, E and 6F). In a younger gynoecium (stage 10) the same pattern is already present (data not shown). It was shown that AG mRNA expression at early gynoecium stages is throughout the whole tissue, but in contrast to our protein localisation data, this expression becomes restricted over time to the stigma and no or hardly any expression exists in the ovary walls in a stage 12 pistil . The high uniform signal of AG:GFP that we observed throughout stage 10 and stage 12 pistils could be explained by a low turnover of the protein molecules in those tissues.
SEP3 and AG localisation correlated with WUSexpression during ovule development
We studied the spatio-temporal localisation pattern of SEP3:GFP and AG:GFP during ovule development in relation to the expression pattern of WUSCHEL (WUS). WUS is a homeodomain transcription factor involved in meristem cell identity maintenance in shoots and an important regulator of ovule development [42, 43]. It is expressed in the centre of the shoot apical meristem, inflorescence and floral meristem and later in the developing ovule [43–45]. The down-regulation of WUS in the terminating floral meristem is thought to be regulated by AG [46, 47]. Additionally, D- and E-type MADS domain proteins like STK and SEP3, might be involved in the suppression of WUS and the termination of the floral meristem  (RI and GA, unpublished results). In ovules, WUS is thought to be down-regulated by the SEP3-AG dimer combined with homeodomain transcription factor BELL1 . For this reason, the localisation pattern of SEP3:GFP and AG:GFP was correlated with the expression pattern of WUS in ovules, which may reveal whether this interaction between these MADS domain proteins and the WUS gene is tightly correlated.
The results described here show that in some cases a discrepancy exists between the previously reported mRNA expression patterns and our protein localisations. Therefore, mRNA expression patterns alone are not sufficient to form hypotheses about gene function and they need to be supported by protein localisation data. For instance, in a recent paper  it was suggested that FUL, unlike AP1, cannot be directly involved in the regulation of flowering time genes (e.g. SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1), simply because it is not expressed in the early flower bud stages. Our localisation data, however, shows that FUL:GFP is present in the early flower bud stages and therefore could have the same function as AP1 in regulating these flowering time genes. This is also in agreement with the hypothesis that both AP1 and FUL act as hubs between the flower induction protein network and the floral organ protein network , and previous genetic studies that showed redundancy between AP1 and FUL during the floral transition . Other examples, such as the asymmetric localisation of SEP3 in the epidermis of both sepals and petals and its possible role in the adaxial/abaxial patterning of these organs, the presence of FUL protein in the replum of developing pistils, or the presence of AG in the nucellus of the developing ovule during the down-regulation of WUS were also not apparent from the reported mRNA patterns. These cases demonstrate the importance of studying protein patterns and protein levels for a better understanding of transcription factor functioning. Some caution is needed however, as we analysed the behaviour of tagged proteins, and we cannot exclude that the localisation patterns of these fusion proteins differ from those of the native proteins. It is possible that the increased size of the fusion protein could have an effect on the transport abilities of the protein, or that the presence of the tag could interfere with the ability to form (multimeric) protein complexes. Furthermore, the presence of the tag could change the stability of the protein, which could lead to a different localisation pattern. We also have to realise that the transgenes are inserted on other positions in the genome than the endogenous genes, which may cause some positional effects on expression pattern and level. Nevertheless, because of the capacity of the AG, FUL and AP1 fusion proteins to complement their respective mutants and the lack of ectopic expression phenotypes in the transgenic lines during floral development, it is very likely that the localisations of the fusion proteins mimic the patterns of the endogenous proteins.
Discrepancies between mRNA and protein patterns may suggest non-cell autonomous action of the proteins by intercellular transport. This has been shown for a number of transcription factors, such as KNOTTED-1 in maize , and SHORTROOT  and CAPRICE  in Arabidopsis. Also MADS domain proteins may move from one cell to another, as has been reported for the Antirrhinum majus MADS domain proteins DEFICIENS and GLOBOSA . In our study there are a few examples where the presence of MADS domain proteins could be due to intercellular transport. For instance, the low level, but distinct presence of SEP3 in the epidermis of the inflorescence meristem and stage 1 flower buds could be due to epidermal transport from high expressing stage 2/3 flower buds. Another example is the increasing AG presence in the nucellus in the developing ovule, where the high expressing integuments might be the source of the AG proteins in the nucellus. Also, as previously suggested , the FUL proteins in the replum in developing pistils could originate from the high expressing valve cells.
Subcellular localisations of proteins can provide clues on the regulation and functioning of transcription factors. The subcellular SEP3:GFP localisation in the cytoplasm in the area of the floral meristem that will become whorl 2 and 3 is an example of a localisation that is not expected based on the nature of transcription factors. The cytoplasmic localisation could indicate that the SEP3:GFP protein in the cytoplasm is not in a dimeric form, and therefore cannot be transported to the nucleus [11–13, 24]. Alternatively, it could also be that post-translational modifications cause the cytoplasmic retention of the SEP3:GFP protein, possibly facilitating intercellular transport  or breakdown of the protein . Recently, it has also been shown that the MADS domain protein MPF2 from Physalis floridana is located in the cytoplasm and imported into the nucleus upon hormone treatment with cytokinin . This shows that non-nuclear localisation is an intriguing mechanism for the regulation of transcription factor functioning.
Summarising, our analysis with GFP tagged proteins expressed under the control of the endogenous promoter revealed the spatio-temporal dynamics of the MADS domain proteins in various tissues of the living plant, leading to a deeper understanding of the behaviour of these MADS domain proteins and allowing the formation of new hypotheses about their function and regulation during early and later stages of floral development.
All plants were grown at 22°C in growth chambers under a long-day light regime (16 h light/8 h dark).
The construction of transgenic lines expressing gAG:GFP, gFUL:GFP and gSEP3:GFP was previously described . The AG genomic clone has a promoter region of approximately 2.6 kb upstream from the translational start, the FUL genomic clone has a promoter region of approximately 2 kb and the SEP3 clone has a promoter region of approximately 1.5 kb. To make the translational gAP1:GFP fusion construct, a genomic clone fragment of AP1 (6616 bp) was amplified with the following two gene specific primers: the forward primer PDS298 (5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTGTTTAACATCCAAGATTTGTTTTACATAATCGTTAC-3') located 2992 bp upstream from the translational start, and the reverse primer PDS297 (5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTGCGGCGAAGCAGCCAAGGTT-3') lacking the stop codon at the 3' end of the coding sequence. The amplified product was inserted into the pENTR/D-TOPO vector (Invitrogen) and, after sequence controls, recombined into the binary vector pMDC107 . Arabidopsis plants were transformed with Agrobacterium tumefaciens strain GV3101 using the floral dip method . Transformed seeds were selected on LB agar plates with 10 μg/ml hygromycin, and stable transgenic lines were maintained afterwards. Furthermore, a transcriptional pWUSCHEL:GUS line  was used to analyse the expression pattern of WUSCHEL in developing ovules.
Stable transgenic plant lines expressing gAG:GFP and gFUL:GFP in wild type Col-0 background were crossed with the SALK_014999 ag T-DNA insertion mutant line and the ful-1 mutant  respectively, while the gAP1:GFP construct was directly transformed into the SALK_056708 ap1 T-DNA insertion mutant line. In the progeny, the presence of the wild type allele, the mutant allele, and the GFP tagged MADS box gene construct was determined by PCR and CLSM. For AG the following primer pairs were used: AG wild type allele, forward primer PRO182 (5'-GGATCCATGGCGTACCAATCGGAGCT-3') annealing immediately after the START codon and reverse primer PDS1985 (5'-CATTTCCTTCAGCCTATATTACC-3') located in the 3' UTR 18 bp downstream of the STOP codon; ag T-DNA mutant allele, forward primer PRO433 (5'-CACCGATCAAAGACTACACATCAC-3') located in the 5' UTR 2634 bp upstream of the START codon and reverse primer PDS404 (5'-TGGTTCACGTAGTGGGCCATCG-3') located on the left border of the T-DNA. The presence of gAG:GFP was determined by CLSM in the inflorescences. For FUL, the following primer pairs were used: FUL wild type allele, forward primer PDS1024 (5'-CTTACGTCACTGTAGACTCACG-3') located in the 5' UTR 201 bp upstream of the START codon and reverse primer PDS1023 (5'-AAAGAGTGAGATAGTTCTACTCG-3') in the 3' UTR 16 bp downstream of the STOP codon; ful-1 mutant allele, forward primer PDS1025 (5'-TTCATCCCTTTTTCAGGGTTGTC-3') corresponding with the inserted DsE element and reverse primer PDS1023; and gFUL:GFP, forward primer PDS920 (5'-ATCACTTACGTCACTGTAGACTCACG-3') in the 5' UTR 204 bp upstream and reverse primer PDS914 (5'-CATCATGTTTGTATAGTTCATCCATGCC-3') 5 bp upstream of the STOP codon of mGFP6. For AP1, the following primer pairs were used: AP1 wild type allele, forward primer PDS912 (5'-AAAACTTTAGGGCCGTAGTGAAGTGAAC-3') 385 bp downstream of the START codon and reverse primer PDS1105 (5'-ATTGGATGAAAAGAGCCTAGCCAC-3') in the 3' UTR 89 bp downstream of the STOP codon (which will give no product for the ap1 T-DNA mutant allele); gAP1:GFP, forward primer PDS912 and reverse primer PDS915 (5'-GACCAGGGTTGGCCATGGAACAGG-3') 183 bp downstream of the START codon of mGFP6.
Confocal laser scanning microscopy
To observe the localisation of the GFP tagged proteins in living plant tissue, inflorescence material was dissected until the relevant meristems and flower buds became visible. After stage 5 flower buds, when sepals started to enclose the floral meristem, it became dificult to visualise the underlying developing floral organs without dissecting the flower buds. The tissues were embedded as previously described . The dye FM4-64 (Molecular Probes, Leiden, The Netherlands) was used as a red counter stain for cell membranes and added at a concentration of 5 μM to the embedding mixture of 0.8% agar, 0.5 × MS. The incubation time of the sample in the embedding mixture with FM4-64 was at least 20 minutes. Confocal Laser Scanning Microscopy (CLSM) of the living plant tissue was performed with a Zeiss LSM 510 inverted confocal microscope using a 40 × C-Apochromat (NA 1.2 W korr) lens. Both GFP and the FM4-64 dye were excited with the 488 nm line of an Argon ion laser. The GFP emission was filtered with a 505–530 nm band pass filter, while the FM4-64 dye emission and red autofluorescence was filtered with a 650 nm long pass filter. The optical slices in the confocal z-stacks were made as a sum of 4 scans and were median filtered afterwards. Three-dimensional projections of the obtained confocal z-stacks were made with the Zeiss LSM Image Browser version 4 and adjusted with Adobe Photoshop version 5.0.
To analyse the expression pattern of WUSCHEL in developing ovules, inflorescences of the pWUS:GUS line were fixed and a β-glucuronidase (GUS) assay was performed overnight at 37°C as previously described  (modified from ). After GUS detection and chlorophyll removal, the inflorescences were kept in Hoyer's solution (7.5 g Arabic gum, 100 g chloral hydrate, 5 ml glycerol, and 60 ml water). Whole siliques of one inflorescence were put under a cover slip and observed with a Nikon Optiphot microscope. Bright field images of the ovules were taken with a Leica DFC320 digital camera using a 40× Plan DIC objective.
We are grateful to Thomas Laux for providing pWUS:GUS seeds. This work was in part financially supported by the Dutch Ministry of Agriculture, Nature management and Food quality and the Centre for BioSystems Genomics (CBSG).
- Riechmann JL, Meyerowitz EM: MADS domain proteins in plant development. Biological Chemistry. 1997, 378 (10): 1079-1101.PubMedGoogle Scholar
- Coen ES, Meyerowitz EM: The war of the whorls – Genetic interactions controlling flower development. Nature. 1991, 353 (6339): 31-37. 10.1038/353031a0.PubMedView ArticleGoogle Scholar
- Colombo L, Franken J, Koetje E, Vanwent J, Dons HJM, Angenent GC, Vantunen AJ: The Petunia MADS box gene FBP11 determines ovule identity. Plant Cell. 1995, 7 (11): 1859-1868. 10.1105/tpc.7.11.1859.PubMedPubMed CentralView ArticleGoogle Scholar
- Pelaz S, Ditta GS, Baumann E, Wisman E, Yanofsky MF: B and C floral organ identity functions require SEPALLATA MADS-box genes. Nature. 2000, 405 (6783): 200-203. 10.1038/35012103.PubMedView ArticleGoogle Scholar
- Pinyopich A, Ditta GS, Savidge B, Liljegren SJ, Baumann E, Wisman E, Yanofsky MF: Assessing the redundancy of MADS-box genes during carpel and ovule development. Nature. 2003, 424 (6944): 85-88. 10.1038/nature01741.PubMedView ArticleGoogle Scholar
- Ditta G, Pinyopich A, Robles P, Pelaz S, Yanofsky MF: The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity. Current Biology. 2004, 14 (21): 1935-1940. 10.1016/j.cub.2004.10.028.PubMedView ArticleGoogle Scholar
- Ferrario S, Immink RG, Angenent GC: Conservation and diversity in flower land. Current Opinion in Plant Biology. 2004, 7 (1): 84-91. 10.1016/j.pbi.2003.11.003.PubMedView ArticleGoogle Scholar
- Mizukami Y, Ma H: Separation of AG function in floral meristem determinacy from that in reproductive organ identity by expressing antisense AG RNA. Plant Molecular Biology. 1995, 28 (5): 767-784. 10.1007/BF00042064.PubMedView ArticleGoogle Scholar
- Ferrandiz C, Gu Q, Martienssen R, Yanofsky MF: Redundant regulation of meristem identity and plant architecture by FRUITFULL, APETALA1 and CAULIFLOWER. Development. 2000, 127 (4): 725-734.PubMedGoogle Scholar
- Mandel MA, Yanofsky MF: A gene triggering flower formation in Arabidopsis. Nature. 1995, 377 (6549): 522-524. 10.1038/377522a0.PubMedView ArticleGoogle Scholar
- McGonigle B, Bouhidel K, Irish VF: Nuclear localization of the Arabidopsis APETALA3 and PISTILLATA homeotic gene products depends on their simultaneous expression. Genes Dev. 1996, 10 (14): 1812-1821. 10.1101/gad.10.14.1812.PubMedView ArticleGoogle Scholar
- Immink RG, Gadella TW, Ferrario S, Busscher M, Angenent GC: Analysis of MADS box protein-protein interactions in living plant cells. Proc Natl Acad Sci USA. 2002, 99 (4): 2416-2421. 10.1073/pnas.042677699.PubMedPubMed CentralView ArticleGoogle Scholar
- Bemer M, Wolters-Arts M, Grossniklaus U, Angenent GC: The MADS domain protein DIANA acts together with AGAMOUS-LIKE80 to specify the central cell in Arabidopsis ovules. Plant Cell. 2008Google Scholar
- Schwarz-Sommer Z, Hue I, Huijser P, Flor PJ, Hansen R, Tetens F, Lonnig WE, Saedler H, Sommer H: Characterization of the Antirrhinum floral homeotic MADS-Box gene DEFICIENS – Evidence for DNA-binding and autoregulation of its persistent expression throughout flower development. Embo Journal. 1992, 11 (1): 251-263.PubMedPubMed CentralGoogle Scholar
- Egea-Cortines M, Saedler H, Sommer H: Ternary complex formation between the MADS-box proteins SQUAMOSA, DEFICIENS and GLOBOSA is involved in the control of floral architecture in Antirrhinum majus. Embo Journal. 1999, 18 (19): 5370-5379. 10.1093/emboj/18.19.5370.PubMedPubMed CentralView ArticleGoogle Scholar
- Honma T, Goto K: Complexes of MADS-box proteins are sufficient to convert leaves into floral organs. Nature. 2001, 409 (6819): 525-529. 10.1038/35054083.PubMedView ArticleGoogle Scholar
- Theissen G, Saedler H: Plant biology – Floral quartets. Nature. 2001, 409 (6819): 469-471. 10.1038/35054172.PubMedView ArticleGoogle Scholar
- de Folter S, Immink RGH, Kieffer M, Parenicova L, Henz SR, Weigel D, Busscher M, Kooiker M, Colombo L, Kater MM, et al: Comprehensive interaction map of the Arabidopsis MADS box transcription factors. Plant Cell. 2005, 17 (5): 1424-1433. 10.1105/tpc.105.031831.PubMedPubMed CentralView ArticleGoogle Scholar
- Bowman JL, Drews GN, Meyerowitz EM: Expression of the Arabidopsis floral homeotic gene AGAMOUS is restricted to specific cell-types late in flower development. Plant Cell. 1991, 3 (8): 749-758. 10.1105/tpc.3.8.749.PubMedPubMed CentralView ArticleGoogle Scholar
- Mandel MA, Yanofsky MF: The Arabidopsis AGL9 MADS box gene is expressed in young flower primordia. Sexual Plant Reproduction. 1998, 11 (1): 22-28. 10.1007/s004970050116.View ArticleGoogle Scholar
- Mandel MA, Gustafsonbrown C, Savidge B, Yanofsky MF: Molecular characterization of the Arabidopsis floral homeotic gene APETALA1. Nature. 1992, 360 (6401): 273-277. 10.1038/360273a0.PubMedView ArticleGoogle Scholar
- Gu Q, Ferrandiz C, Yanofsky MF, Martienssen R: The FRUITFULL MADS-box gene mediates cell differentiation during Arabidopsis fruit development. Development. 1998, 125 (8): 1509-1517.PubMedGoogle Scholar
- Perbal MC, Haughn G, Saedler H, SchwarzSommer Z: Non-cell-autonomous function of the Antirrhinum floral homeotic proteins DEFICIENS and GLOBOSA is exerted by their polar cell-to-cell trafficking. Development. 1996, 122 (11): 3433-3441.PubMedGoogle Scholar
- Ferrario S, Busscher J, Franken J, Gerats T, Vandenbussche M, Angenent GC, Immink RGH: Ectopic expression of the petunia MADS box gene UNSHAVEN accelerates flowering and confers leaf-like characteristics to floral organs in a dominant-negative manner. Plant Cell. 2004, 16 (6): 1490-1505. 10.1105/tpc.019679.PubMedPubMed CentralView ArticleGoogle Scholar
- Haseloff J, Dorman E-L, Brand AH: Live imaging with green fluorescent protein. 1999, Humana Press, 122:Google Scholar
- Sheen J, Hwang SB, Niwa Y, Kobayashi H, Galbraith DW: Green fluorescent protein as a new vital marker in plant-cells. Plant Journal. 1995, 8 (5): 777-784. 10.1046/j.1365-313X.1995.08050777.x.PubMedView ArticleGoogle Scholar
- Shu XK, Shaner NC, Yarbrough CA, Tsien RY, Remington SJ: Novel chromophores and buried charges control color in mFruits. Biochemistry. 2006, 45 (32): 9639-9647. 10.1021/bi060773l.PubMedView ArticleGoogle Scholar
- Wu XL, Dinneny JR, Crawford KM, Rhee Y, Citovsky V, Zambryski PC, Weigel D: Modes of intercellular transcription factor movement in the Arabidopsis apex. Development. 2003, 130 (16): 3735-3745. 10.1242/dev.00577.PubMedView ArticleGoogle Scholar
- de Folter S, Urbanus SL, van Zuijlen LG, Kaufmann K, Angenent GC: Tagging of MADS domain proteins for chromatin immunoprecipitation. BMC Plant Biol. 2007, 7 (1): 47-10.1186/1471-2229-7-47.PubMedPubMed CentralView ArticleGoogle Scholar
- Sieburth LE, Meyerowitz EM: Molecular dissection of the AGAMOUS control region shows that cis elements for spatial regulation are located intragenically. Plant Cell. 1997, 9 (3): 355-365. 10.1105/tpc.9.3.355.PubMedPubMed CentralView ArticleGoogle Scholar
- Kooiker M, Airoldi CA, Losa A, Manzotti PS, Finzi L, Kater MM, Colombo L: BASIC PENTACYSTEINE1, a GA binding protein that induces conformational changes in the regulatory region of the homeotic arabidopsis gene SEEDSTICK. Plant Cell. 2005, 17 (3): 722-729. 10.1105/tpc.104.030130.PubMedPubMed CentralView ArticleGoogle Scholar
- Jack T, Brockman LL, Meyerowitz EM: The homeotic gene APETALA3 of Arabidopsis thaliana encodes a MADS box and is expressed in petals and stamens. Cell. 1992, 68 (4): 683-697. 10.1016/0092-8674(92)90144-2.PubMedView ArticleGoogle Scholar
- Chytilova E, Macas J, Galbraith DW: Green fluorescent protein targeted to the nucleus, a transgenic phenotype useful for studies in plant biology. Annals of Botany. 1999, 83 (6): 645-654. 10.1006/anbo.1999.0866.View ArticleGoogle Scholar
- Yanofsky MF, Ma H, Bowman JL, Drews GN, Feldmann KA, Meyerowitz EM: The protein encoded by the Arabidopsis homeotic gene AGAMOUS resembles transcription factors. Nature. 1990, 346 (6279): 35-39. 10.1038/346035a0.PubMedView ArticleGoogle Scholar
- Drews GN, Bowman JL, Meyerowitz EM: Negative regulation of the Arabidopsis homeotic gene AGAMOUS by the APETALA2 product. Cell. 1991, 65 (6): 991-1002. 10.1016/0092-8674(91)90551-9.PubMedView ArticleGoogle Scholar
- Mandel MA, Yanofsky MF: The Arabidopsis AGL8 MADS box gene is expressed in inflorescence meristems and is negatively regulated by APETALA1. Plant Cell. 1995, 7 (11): 1763-1771. 10.1105/tpc.7.11.1763.PubMedPubMed CentralView ArticleGoogle Scholar
- Gustafson-Brown C, Savidge B, Yanofsky MF: Regulation of the Arabidopsis floral homeotic gene APETALA1. Cell. 1994, 76 (1): 131-143. 10.1016/0092-8674(94)90178-3.PubMedView ArticleGoogle Scholar
- Ito T, Ng KH, Lim TS, Yu H, Meyerowitz EM: The homeotic protein AGAMOUS controls late stamen development by regulating a jasmonate biosynthetic gene in Arabidopsis. Plant Cell. 2007, 19 (11): 3516-3529. 10.1105/tpc.107.055467.PubMedPubMed CentralView ArticleGoogle Scholar
- Liljegren SJ, Roeder AHK, Kempin SA, Gremski K, Ostergaard L, Guimil S, Reyes DK, Yanofsky MF: Control of fruit patterning in Arabidopsis by INDEHISCENT. Cell. 2004, 116 (6): 843-853. 10.1016/S0092-8674(04)00217-X.PubMedView ArticleGoogle Scholar
- Smyth DR, Bowman JL, Meyerowitz EM: Early flower development in Arabidopsis. Plant Cell. 1990, 2 (8): 755-767. 10.1105/tpc.2.8.755.PubMedPubMed CentralView ArticleGoogle Scholar
- Ferrandiz C, Liljegren SJ, Yanofsky MF: Negative regulation of the SHATTERPROOF genes by FRUITFULL during Arabidopsis fruit development. Science. 2000, 289 (5478): 436-438. 10.1126/science.289.5478.436.PubMedView ArticleGoogle Scholar
- Laux T, Mayer KFX, Berger J, Jurgens G: The WUSCHEL gene is required for shoot and floral meristem integrity in Arabidopsis. Development. 1996, 122 (1): 87-96.PubMedGoogle Scholar
- Gross-Hardt R, Lenhard M, Laux T: WUSCHEL signaling functions in interregional communication during Arabidopsis ovule development. Genes & Development. 2002, 16 (9): 1129-1138. 10.1101/gad.225202.View ArticleGoogle Scholar
- Mayer KFX, Schoof H, Haecker A, Lenhard M, Jurgens G, Laux T: Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot meristem. Cell. 1998, 95 (6): 805-815. 10.1016/S0092-8674(00)81703-1.PubMedView ArticleGoogle Scholar
- Schoof H, Lenhard M, Haecker A, Mayer KFX, Jurgens G, Laux T: The stem cell population of Arabidopsis shoot meristems is maintained by a regulatory loop between the CLAVATA and WUSCHEL genes. Cell. 2000, 100 (6): 635-644. 10.1016/S0092-8674(00)80700-X.PubMedView ArticleGoogle Scholar
- Lenhard M, Bohnert A, Jurgens G, Laux T: Termination of stem cell maintenance in Arabidopsis floral meristems by interactions between WUSCHEL and AGAMOUS. Cell. 2001, 105 (6): 805-814. 10.1016/S0092-8674(01)00390-7.PubMedView ArticleGoogle Scholar
- Lohmann JU, Hong RL, Hobe M, Busch MA, Parcy F, Simon R, Weigel D: A molecular link between stem cell regulation and floral patterning in Arabidopsis. Cell. 2001, 105 (6): 793-803. 10.1016/S0092-8674(01)00384-1.PubMedView ArticleGoogle Scholar
- Ferrario S, Shchennikova AV, Franken J, Immink RGH, Angenent GC: Control of floral meristem determinacy in petunia by MADS-box transcription factors. Plant Physiology. 2006, 140 (3): 890-898. 10.1104/pp.105.072660.PubMedPubMed CentralView ArticleGoogle Scholar
- Brambilla V, Battaglia R, Colombo M, Masiero S, Bencivenga S, Kater MM, Colombo L: Genetic and molecular interactions between BELL1 and MADS box factors support ovule development in Arabidopsis. Plant Cell. 2007, 19 (8): 2544-2556. 10.1105/tpc.107.051797.PubMedPubMed CentralView ArticleGoogle Scholar
- Schneitz K, Hulskamp M, Pruitt RE: Wild-type ovule development in Arabidopsis thaliana – A light-microscope study of cleared whole-mount tissue. Plant Journal. 1995, 7 (5): 731-749. 10.1046/j.1365-313X.1995.07050731.x.View ArticleGoogle Scholar
- Reiser L, Modrusan Z, Margossian L, Samach A, Ohad N, Haughn GW, Fischer RL: The BELL1 gene encodes a homeodomain protein involved in pattern-formation in the Arabidopsis ovule primordium. Cell. 1995, 83 (5): 735-742. 10.1016/0092-8674(95)90186-8.PubMedView ArticleGoogle Scholar
- Liu C, Zhou J, Bracha-Drori K, Yalovsky S, Ito T, Yu H: Specification of Arabidopsis floral meristem identity by repression of flowering time genes. Development. 2007, 134 (10): 1901-1910. 10.1242/dev.003103.PubMedView ArticleGoogle Scholar
- Jackson D: Double labeling of KNOTTED1 mRNA and protein reveals multiple potential sites of protein trafficking in the shoot apex. Plant Physiology. 2002, 129 (4): 1423-1429. 10.1104/pp.006049.PubMedPubMed CentralView ArticleGoogle Scholar
- Nakajima K, Sena G, Nawy T, Benfey PN: Intercellular movement of the putative transcription factor SHR in root patterning. Nature. 2001, 413 (6853): 307-311. 10.1038/35095061.PubMedView ArticleGoogle Scholar
- Wada T, Kurata T, Tominaga R, Koshino-Kimura Y, Tachibana T, Goto K, Marks MD, Shimura Y, Okada K: Role of a positive regulator of root hair development, CAPRICE, in Arabidopsis root epidermal cell differentiation. Development. 2002, 129 (23): 5409-5419. 10.1242/dev.00111.PubMedView ArticleGoogle Scholar
- Lucas WJ, Lee JY: Plant cell biology – Plasmodesmata as a supracellular control network in plants. Nature Reviews Molecular Cell Biology. 2004, 5 (9): 712-726. 10.1038/nrm1470.PubMedView ArticleGoogle Scholar
- Smalle J, Vierstra RD: The ubiquitin 26S proteasome proteolytic pathway. Annual Review of Plant Biology. 2004, 55: 555-590. 10.1146/annurev.arplant.55.031903.141801.PubMedView ArticleGoogle Scholar
- He CY, Saedler H: Hormonal control of the inflated calyx syndrome, a morphological novelty, in Physalis. Plant Journal. 2007, 49 (5): 935-946. 10.1111/j.1365-313X.2006.03008.x.PubMedView ArticleGoogle Scholar
- Curtis MD, Grossniklaus U: A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiology. 2003, 133 (2): 462-469. 10.1104/pp.103.027979.PubMedPubMed CentralView ArticleGoogle Scholar
- Clough SJ, Bent AF: Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal. 1998, 16 (6): 735-743. 10.1046/j.1365-313x.1998.00343.x.PubMedView ArticleGoogle Scholar
- de Folter S, Shchennikova AV, Franken J, Busscher M, Baskar R, Grossniklaus U, Angenent GC, Immink RGH: A B-sister MADS-box gene involved in ovule and seed development in petunia and Arabidopsis. Plant Journal. 2006, 47 (6): 934-946. 10.1111/j.1365-313X.2006.02846.x.PubMedView ArticleGoogle Scholar
- Bade J, van Grinsven E, Custers J, Hoekstra S, Ponstein A: T-DNA tagging in Brassica napus as an efficient tool for the isolation of new promoters for selectable marker genes. Plant Molecular Biology. 2003, 52 (1): 53-68. 10.1023/A:1023980326336.PubMedView ArticleGoogle Scholar