Current Research
 
Duplications in the ABC genes at the base of the core eudicots
 
Researcher: Cheng-Chiang Wu
 
    The family of plant transcription factors known as the MADS-box genes has received a great deal of attention, both in terms of function and evolution.  The super-family of MADS-box containing genes includes representatives from yeast (MCM1) and animals (SRF), however the family has diversified extensively via numerous duplication events within plants.  Their common feature is a conserved 60-amino acid region, the MADS domain, which has been shown to bind DNA in a sequence specific manner and contribute to the proteins’ ability to function as dimers.  Perhaps not surprisingly, MADS-domain proteins from yeast, animals and plants display overlapping binding specificities, recognizing DNA elements called CArG boxes.  Many plant MADS-box genes display additional highly conserved modules, conforming to what is known as the MIKC structure (above). Early in the study of plant MADS-box genes, a second conserved domain was recognized, the K-box, which has loose sequence similarity to the coiled-coil domain of keratin.  This region of approximately 70 aa facilitates the dimerization of MIKC-type MADS-domain proteins.  The stretch of ca. 30 aa located between the MADS and K domains, referred to as the Intervening (I) region, also appears to be critical for the selective formation of dimers.  Although the C-terminal domain is quite variable across the family, it does contain highly conserved, lineage-specific motifs and has recently been implicated as a mediator of “quartet” complex formation between dimers of MIKC-type MADS-domain proteins.  The association of different dimer combinations in these higher-order protein complexes may affect the DNA-binding specificity of the subunits and, in turn, produce different morphologies.  In addition, the capacity of some MIKC-type genes to promote transcriptional activation been mapped to the C-terminal region (see Becker and Theissen, 2003 and Jack, 2004 for recent reviews of MADS box gene function and evolution).
 
    The discovery that MIKC genes play many fundamental roles in floral development have spurred great interest in the plant MADS-box gene family, leading to the discovery of many more representatives in Arabidopsis and several other model species including Antirrhinum, Petunia, and Nicotiana.  Phylogenetic analyses of these genes have shown that the family is made up of several subfamilies whose members typically have similar functions.  For instance, the Arabidopsis B class genes, APETALA3 (AP3, the ortholog of DEF), and PISTILLATA (PI), each define their own orthologous lineages which are themselves closely related paralogous lineages, thereby defining a clade of B function genes.  The various subfamilies are not static, however, showing evidence of multiple duplication events that have given rise to paralogous lineages often possessing unique functional repertoires.  Of particular relevance to this project are a coincident set of gene duplication events that occurred in the APETALA1 (AP1), AP3, AG and SEP1/2/4 gene lineages.  These duplications appear to map close to the base of the angiosperm core eudicot clade and are notable for a number of reasons. First, they affect one major lineage from each of the four main classes of organ identity genes: AP1 in the A class, AP3 in the B class, AG in the C class and SEP1/2/4 in the E class (reviewed Kramer and Zimmer, 2006). Second, in the AP1 and AP3 lineages the duplications are followed by striking patterns of sequence divergence.  Both of these Arabidopsis genes are actually representatives of divergent paralogous lineages and exhibit sequence motifs that are entirely distinct from the ancestral forms, a phenomenon that is most apparent in the C-terminal motifs (Kramer et al., 1998; Litt and Irish, 2003).  In the case of AP1, a duplication in the ancestral FUL-like lineage produced the euAP1 and euFUL lineages, which are represented in Arabidopsis by AP1 and FRUITFULL (FUL), respectively.  These lineages are easily distinguished by their distinct C-terminal motifs: the ancestral FUL-like and euFUL members all have FUL-like motifs while the euAP1 orthologs have euAP1 motifs.  Similarly, there are two core eudicot lineages of AP3-like genes known as euAP3 (including AP3) and TM6, which does not have an Arabidopsis representative. These paralogs were derived from a duplication in the ancestral paleoAP3 lineage.  We see that the ancestral paleoAP3 C-terminal motif is conserved throughout the paleoAP3 and TM6 lineages but orthologs of euAP3 display distinctly different euAP3 motifs.  In contrast, although the ancestral AG-like lineage was duplicated in the lower eudicots, giving rise to the euAG and PLE lineages, these paralogs did not experience significant sequence divergence (Kramer et al., 2004).  Similarly, the duplications in the SEP1/2/4 lineage are not associated with major sequence remodeling (Zahn et al., 2005).  The sequence divergence that produced the derived euAP1 and euAP3 motifs is notable in that it involved dramatic remodeling due to frameshift mutations, as well as more typical nonsynonymous changes (Litt and Irish, 2003; Vandenbussche et al., 2003; Kramer et al., 2006).  In addition to these interesting molecular evolutionary phenomena, these duplications appear to be correlated with changes in aspects of biochemical function and gene regulation (Lamb and Irish, 2003; reviewed Kramer, 2005 and Kramer and Zimmer, 2006).  Furthermore, the timing of the three duplications in the AP1, AP3, AG and SEP lineages is associated with the emergence of the core eudicot clade, which is the largest and most successful angiosperm lineage.
 
    We believe that the duplications in the lower eudicot AP1, AP3 and AG lineages represent a unique opportunity to study the relationship between gene duplication and the diversification of gene function during organismal evolution.  The coincident duplication of three key gene lineages, along with the evolution of fixed floral architecture, allow us to combine extensive knowledge of gene lineage evolution, angiosperm phylogeny, and floral morphology to produce a comprehensive picture of the evolutionary events that occurred at this juncture.  In collaboration with Dr. Amy Litt of the NY Botanical Garaden and Dr. Jer-Ming Hu of National Taiwan University, we are pursuing several type of analyses that will provide specific information regarding the changes that occurred following the duplications in these three key gene lineages, and the effect these changes had on gene function.  These include a thorough phylogenetic analysis of AP3, AG and AP1 lineage evolution in all the lineages of the lower eudicots, comparative studies of gene expression, characterization of protein interactions across diverse taxa, and heterologous expression analyses.
 
Becker, A., and Theissen, G. (2003). The major clades of MADS-box genes and their role
    in the development and evolution of flowering plants. Mol Phy Evol 29, 464-489.
Jack, T. (2004). Molecular and genetic mechanisms of floral control. Plant Cell 16, S1-
    S17.
Litt, A., and Irish, V.F. (2003). Duplication and diversification in the
APETALA1/FRUITFULL floral homeotic gene lineage: implications for the evolution of floral development. Genetics 165, 821-833.
Kramer, E.M. (2005). Floral architecture: Regulation and diversity of floral shape and pattern. In Plant architecture and its manipulation., C.G.N. Turnbull, ed (Oxford, UK: Blackwell Publishing), pp. 120-147.
Kramer, E.M., Dorit, R.L., and Irish, V.F. (1998). Molecular evolution of genes controlling petal and stamen development: Duplication and divergence within the APETALA3 and PISTILLATA MADS-box gene lineages. Genetics 149, 765-783.
Kramer, E. M., Su, H.-J., Wu, C.-C., and  Hu, J.-M. (2006) A simplified explanation for the
        frameshift mutation that created a novel C-terminal motif in the APETALA3 gene lineage.
Kramer, E.M., Jaramillo, M.A., and Di Stilio, V.S. (2004). Patterns of gene duplication and functional evolution during the diversification of the AGAMOUS subfamily of MADS-box genes in angiosperms. Genetics 166, 1011-1023.
Kramer, E. M. and Zimmer, E. A. (2006) Gene duplication and floral developmental genetics of
        basal eudicots. Pp. 352-381 in P. Soltis, D. Soltis, J. Leebens-Mack, ed. Advances in Botanical
        Research, vol. 44.
Lamb, R.S., and Irish, V.F. (2003). Functional divergence within the
    APETALA3/PISTILLATA floral homeotic gene lineages. Proc Nat’l Acad Sci
    USA 100, 6558-6563.
Vandenbussche, M., Theissen, G., Van de Peer, Y., and Gerats, T. (2003). Structural
        diversification and neo-functionalization during floral MADS-box gene evolution by C-    
        terminal frameshift mutations. NAR 31, 4401-4409.
Zahn, L.M., Kong, H., Leebens-Mack, J.H., Kim, S., Soltis, P.M., Landherr, L.L., Soltis, D.E.,
        dePamphilis, C.W. and Ma, H. (2005). The evolution of the SEPALLATA subfamily of
        MADS-Box genes: A preangiosperm origin with multiple duplications throughout
        angiosperm history. Genetics 169, 2209-2223.