Protein interactions of MADS box transcription factors involved in flowering in Lolium perenne.

Regulation of flowering time is best understood in the dicot model species Arabidopsis thaliana. Molecular analyses revealed that genes belonging to the MADS box transcription factor family play pivotal regulatory roles in both the vernalization- and photoperiod-regulated flowering pathways. Here the analysis of three APETALA1 (AP1)-like MADS box proteins (LpMADS1-3) and a SHORT VEGETATIVE PHASE (SVP)-like MADS box protein (LpMADS10) from the monocot perennial grass species Lolium perenne is reported. Features of these MADS box proteins were studied by yeast two-hybrid assays. Protein-protein interactions among the Lolium proteins and with members of the Arabidopsis MADS box family have been studied. The expression pattern for LpMADS1 and the protein properties suggest that not the Arabidopsis AP1 gene, but the SUPPRESSOR OF CONSTANS1 (SOC1) gene, is the functional equivalent of LpMADS1. To obtain insight into the molecular mechanism underlying the regulation of LpMADS1 gene expression in vernalization-sensitive and -insensitive Lolium accessions, the upstream sequences of this gene from a winter and spring growth habit variety were compared with respect to MADS box protein binding. In both promoter elements, a putative MADS box transcription factor-binding site (CArG-box) is present; however, the putative spring promoter has a short deletion adjacent to this DNA motif. Experiments using yeast one-hybrid and gel retardation assays demonstrated that the promoter element is bound by an LpMADS1-LpMADS10 higher order protein complex and, furthermore, that this complex binds efficiently to the promoter element from the winter variety only. This strongly supports the model that LpMADS1 together with LpMADS10 controls the vernalization-dependent regulation of the LpMADS1 gene, which is part of the vernalization-induced flowering process in Lolium.

In vivo imaging of MADS-box transcription factor interactions.

MADS-box transcription factors are major regulators of development in flowering plants. The factors act in a combinatorial manner, either as homo- or heterodimers, and they control floral organ formation and identity and many other developmental processes through a complex network of protein-protein and protein-DNA interactions. Despite the fact that many studies have been carried out to elucidate MADS-box protein dimerization by yeast systems, very little information is available on the behaviour of these molecules in planta. Here, evidence for specific interactions between the petunia MADS-box proteins FBP2, FBP11, and FBP24 is provided in vivo. The dimers identified in yeast for the ovule-specific FBP24 protein have been confirmed in living plant cells by means of fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy and, in addition, some of the most likely, less stable homo- and heterodimers were identified. This in vivo approach revealed that particular dimers could only be detected in specific sub-nuclear domains. In addition, evidence for the in planta assembly of these ovule-specific MADS-box transcription factors into higher-order complexes is provided.