Dimerization specificity of Arabidopsis MADS domain homeotic proteins APETALA1, APETALA3, PISTILLATA, and AGAMOUS.

Abstract

The MADS domain homeotic proteins APETALA1 (API), APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG) act in a combinatorial manner to specify the identity ofArabidopsis floral organs. The molecular basis for this combinatorial mode of action was investigated.Immunoprecipitation experiments indicate that all four proteins are capable of interacting with each other. However, these proteins exhibit "partner-specificity" for the formation of DNA-binding The study of homeotic mutants in Arabidopsis thaliana and Antirrhinum majus has led to the establishment of a genetic model (the ABC model) that explains how the fates of floral organ primordia are determined (1, 2). According to the ABC model, the identities of the organs of an Arabidopsis flower (four sepals, four petals, six stamens, and two carpels) are specified by the action of at least five organ identity genes, all of which have been cloned: APETALA1 (AP1), APETALA2 (AP2),APETALA3 (AP3), PISTILLATA (PI), andAGAMOUS (AG) (3-7). In situ hybridization and ectopic expression experiments (8-10) have provided strong evidence supporting the ABC model, showing that each whorl of a flower primordium has a unique combination of organ identity activities, which combinatorially specify organ identity. The specification of sepals is dependent on class A gene activities (AP1 and AP2), petals are specified by a combination of class A and class B (AP3 and PI) gene activities, stamens are specified by the combined activities of classes B and C (AG), and specification of the carpels is achieved by class C activity.AP1, AP3, PI, and AG are all MADS domain-containing proteins (Fig. 1), while AP2 bears similarity to a different class of DNA binding proteins (4, 13). The MADS domain is a conserved region of 56 amino acids present in a variety of dimeric transcription factors from different organisms. The MADS regions of SRF and MCM1 have been characterized as DNA binding and dimerization domains (11,14). Within the family of MADS box proteins, the plant proteins are unique in that they contain another conserved region, the K box, that may form amphipathic alpha helices (ref. 12; Fig. 1 Here, we show that AP1, AP3, PI, and AG are all capable of interacting with each other, but that only AP1/AP1, AP3/PI, and AG/AG dimers (and also heterodimers formed by truncated AG and AP1) bind to CArG-box containing sequences. This DNA-binding partner specificity is mediated to a large degree by the L region of these proteins, a 31-35 aa segment located between the MADS and K boxes. MATERIALS AND METHODSPlasmid Constructioh. AP1, AP3, PI, and AG cDNA coding sequences were cloned into the pSPUTK in vitro translation vector (Stratagene). Because the initiating ATG codon was not found in the AG cDNA clones (7), the wild-type sequence 5'-CATTTT ... at the beginning of theAG cDNA was changed to 5'-ATGGGG.... The in vivo functionality of such an altered AG protein has been shown in ectopic expression experiments (8). The construction of PPIFLAG, to make FLAG epitopetagged PI protein, has been ...

The K domain mediates heterodimerization of theArabidopsisfloral organ identity proteins, APETALA3 and PISTILLATA

“…Between the MADS and K domains is an intervening region (the I domain) that varies in length. Domain swapping studies suggest that I domain is necessary for dimerization and functional specificity of the Arabidopsis MADS proteins APETALA3 (AP3), PISTILLATA (PI), AGAMOUS (AG) and APETALA1 (AP1) (Krizek and Meyerowitz, 1996b;Riechmann and Meyerowitz, 1997a;Riechmann et al, 1996a). The C-terminal domain is variable in length and sequence among different plant MADS proteins.…”

APETALA1 and SEPALLATA3 interact to promote flower development

“…MADS-box proteins are composed of four different domains, designated M, I, K and C. The MADS (M) domain is highly conserved among these proteins, and is responsible for binding to DNA in addition to its participation in homodimer formation of some proteins. The I region also participates in homodimer formation Riechmann et al, 1996). Adjacent to the I region is the K domain, so named due to its similarity to the coiled-coil domain of keratin.…”

Non-AUG Initiation of AGAMOUS mRNA Translation in Arabidopsis thaliana

“…It therefore appears likely that AG constructs in which an ATG codon was engineered at the 5Ј end of the cDNA were capable of retaining AG function because, in addition to the artifactual form of AG, the correct AG protein was synthesized from the downstream ACG triplet. These constructs include the pSPUTK-AG plasmid that we have used before (51,52), constructs for AG expression in Escherichia coli and subsequent DNA binding assays (44), as well as constructs for ectopic expression of AG in transgenic plants under the control of the 35S promoter (transgenic lines of the 35S-ATG-1 series [41,43]). In fact, all of those constructs were derived from the observation that 35S-ATG-1 lines showed ectopic AG activity (41), which was interpreted as evidence for the functionality of the AG protein synthesized from the artifactual ATG codon introduced at the 5Ј end of AG cDNA.…”

“…pSPUTK-AG is a pSPUTK (Stratagene)-derived plasmid to produce AG in in vitro transcription-translation reactions (51,52). To construct pSPUTK-AG, the wild-type sequence 5Ј-CATTTT at the beginning of the AG cDNA was changed by means of a PCR amplification to 5Ј-ATGGGG, in order to provide an artificial initiating ATG codon.…”

“…[ 35 S]methionine-labeled proteins were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis on 12.5% gels. The DNA-binding activities of pSPUTK-AG-, pSPUTK-AG ATG1 -, and pSPUTK-AG ATG2 -derived proteins were tested in electrophoretic mobility shift assays, which were performed as described previously (51,52).…”