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IN THIS ISSUE

Functional Divergence of AP3 Genes in the MAD World of Flower Development

neckardt{at}aspb.org

Angiosperms emerged 130 million years ago in the Cretaceous period and have evolved into 260,000 species classified into 453 families (Soltis et al., 2005) based in large part on differences in reproductive characteristics. Not surprisingly, understanding the molecular basis of this diversity rests heavily on understanding the evolution of genes that control floral organ identity. Many of these genes encode MADS domain transcription factors that are thought to interact in a combinatorial fashion in multimeric protein complexes to specify the distinct organs of an inflorescence (Coen and Meyerowitz, 1991; Honma and Goto, 2001; Theissen, 2001). The ABC model proposed by Coen and Meyerowitz (1991) continues to be useful in describing the functions of genes involved in floral organ development. In this model, A-class gene activity specifies sepals in the first (outermost) whorl, A- and B-class genes together specify petals in the second whorl, B- and C-class genes together specify stamens in the third whorl, and C-class genes alone in whorl four specify carpels.

Gene duplication is estimated to occur at a high rate in eukaryotic genomes in general (Lynch and Conery, 2000) and in flowering plants in particular (Blanc and Wolfe, 2004; Cui et al., 2006). It is hypothesized that gene duplication arising through polyploidy (whole-genome duplication) has played an important role in the evolution of diversity in flowering plants (de Bodt et al., 2005; Duarte et al., 2006). Kramer et al. (1998) noted that the evolution of petals in the angiosperms may be reflected in the diversified structure and function of B-group genes, represented in Arabidopsis by APETALA3 (AP3) and PISTILLATA (PI), which represent lineages that arose from a duplication event prior to the evolution of the magnolid dicots. These authors analyzed homologous sequences from a number of angiosperm species and determined that a subsequent duplication event occurred in the paleoAP3 lineage coincident with the base of the core eudicot radiation, giving rise to two major AP3 lineages found in the core eudicots, termed euAP3 and TM6 (for TOMATO MADS BOX GENE6, the first gene in this lineage to be characterized; Pnueli et al., 1991). Kramer et al. (1998) speculated that this duplication event in the paleoAP3 lineage may have led to the evolution of a petal-specific AP3 function in the core eudicot lineage.

The euAP3 and TM6 lineages are distinguished principally by a major difference in the C-terminal region, which is highly conserved within each lineage. The TM6 C-terminal motif is more similar to that of the ancestral paleoAP3 lineage, and the distinct C-terminal motif of the euAP3 lineage genes (which are absent in the magnolid and lower eudicots) appears to have arisen by a frameshift mutation (Vandenbussche et al., 2003). Not all core eudicots contain both euAP3 and TM6 lineages; TM6 homologs are clearly missing from Arabidopsis and possibly also from Antirrhinum. Members of the Solanaceae, including petunia, tomato, and potato, have both euAP3 and TM6 homologs. Thus, Solanaceae species present good models for investigating the putative diversification of gene function within the two lineages.

Duplicated genes are generally considered to adopt one of three possible fates: nonfunctionalization (silencing of one copy), neofunctionalization (acquisition of a novel function for one copy), or subfunctionalization (partitioning of tissue-specific patterns of expression of the ancestral gene between the two copies) (Lynch and Conery, 2000). Lynch and Force (2000) presented theoretical evidence that subfunctionalization may be a common fate of duplicate gene pairs, particularly for genes that contain multiple regulatory regions, because the partitioning of gene expression patterns can result from degenerative loss-of-function mutations, which occur much more frequently than beneficial gain-of-function mutations that might lead to neofunctionalization. It is also worth noting that these two fates are not necessarily mutually exclusive. Indeed, Lynch and Force (2000) suggested that subfunctionalization might facilitate neofunctionalization by preserving gene duplicates and maintaining their exposure to selective forces and/or removing pleiotropic constraints.

In this issue of The Plant Cell, two studies conducted using different members of the Solanaceae present evidence of subfunctionalization of duplicated genes in these AP3 gene lineages. Rijpkema et al. (pages 1819–1832) examined the Petunia hybrida genes TM6 and DEF (the AP3 homolog in petunia), and de Martino et al. (pages 1833–1845) investigated the function of the homologous genes in tomato (Solanum lycopersicum), termed TM6 and TAP3, respectively.

Rijpkema et al. identified the insertional mutant tm6-1 from available Petunia transposon libraries, which likely represents a null mutation in petunia TM6, and also developed transgenic Petunia lines in which TM6 was downregulated by RNA interference. None of these loss-of-function mutants exhibited an abnormal floral phenotype, indicating that petunia TM6 shares function with one or more other genes. The most likely candidate for shared function is DEF, and genetic crosses were performed with petunia def mutants to obtain def tm6 double mutants.

Homozygous def mutants exhibit homeotic conversion of petals to sepals in the second whorl, but stamen development in the third whorl is unaffected. By contrast, the tm6/+def mutant plants showed severely abnormal third whorl organs with a partial loss of determinacy, in addition to the second whorl abnormalities present in def single mutants. Pollen maturation and fertility was severely impaired in tm6/+ def plants, but the occasional viable seed produced several def-1 tm6-1 homozygous double mutants, and these plants exhibited complete homeotic conversion of petals to sepals in the second whorl and of stamens to a fused carpelloid structure in the third whorl. These results indicate that petunia DEF and TM6 act together to determine stamen identity in the third whorl and together fulfill B-class function (petal and stamen identity in second and third whorls, respectively).

The authors next isolated putative 5' regulatory sequences of the two genes in Petunia and also of the orthologs from tomato, TAP3 and TM6, and compared these sequences with other published euAP3 and paleoAP3 5' putative regulatory sequences. These results revealed the presence of distinct and highly conserved domains between the euAP3 and TM6 lineages. Together with the previously described differences in protein–protein interactions and regulation between petunia DEF and TM6 (Vandenbussche et al., 2004), this clearly demonstrates that the two lineages have diverged in function.

de Martino et al. conducted functional analyses of the orthologous tomato genes TAP3 and TM6, identifying and characterizing a loss-of-function transposon insertion in TAP3 and RNA interference–induced loss of function of TM6 (TM6i). Unlike the petunia def mutants, the tomato tap3 homozygous mutant plants exhibited complete transformation of petals into sepaloid structures in the second whorl and stamens into carpelloid structures in the third whorl, indicating that wild-type expression of other genes (such as tomato TM6) are unable to compensate for loss of TAP3 function.

Interestingly, transgenic TM6i tomato plants, which lacked TM6 expression but retained expression of TAP3, also showed a moderate to severely abnormal phenotype, particularly in the third whorl (unlike the corresponding loss-of-function tm6 petunia mutant). In addition, second whorl petals in TM6i tomato plants were reduced in size but did not display homeotic conversions, indicating an effect on cell proliferation. Expression analyses in wild-type tomato showed that TAP3 was expressed at high levels in petals and stamens, whereas TM6 was expressed mainly in stamens and carpels. These results suggest that TAP3 and TM6 have distinct functions in tomato flower development, which do not overlap to the same extent as that of the orthologous genes in petunia.

Somewhat surprisingly, ectopic 35S-driven expression of either tomato TAP3 or TM6 brought about partial restoration of petals in the tap3 mutant. Rijpkema et al. similarly found that overexpression of 35S-driven petunia TM6 brought about partial to complete restoration of petal identity in the petunia def mutant background. These results show that both tomato and petunia TM6 genes retain a capacity for specifying petal development, even though they do not normally appear to exercise this function. In wild-type tomato flowers, TM6 is expressed at lower levels and in a different spatial domain than TAP3, which might account for some of the differences in gene function observed in the mutant analyses. In Arabidopsis, AP3 (euAP3 lineage) is thought to direct stamen development in whorl three in a quaternary complex with PI, AGAMOUS, and SEPALLATA3. Therefore, de Martino et al. tested the ability of tomato TAP3 and TM6 proteins to interact with the proteins encoded by the tomato orthologs of these three genes, TPI, TAG1, and TM5, respectively. Both TAP3 and TM6 interacted strongly with TPI; and TAP3-TPI and TM6-TPI dimers could bind to TM5. However, TAP3 alone was able to bind to TM5, whereas TM6 alone could not. In addition, TAP3 was able to form a quaternary complex in the yeast assay with the other three proteins, whereas TM6 could not, suggesting that tomato TM6 and TAP3 have diverged in their protein–protein interaction capabilities.

The results of both studies are strongly supportive of the hypothesis that gene duplication was followed by subfunctionalization in the AP3 lineage, with euAP3-type genes evolving to play a primary role in petal and stamen development, whereas TM6-type genes have a partially overlapping function in stamen development. As noted above, Kramer et al. (1998) suggested this gene duplication event may have allowed for the evolution of novel gene function in the euAP3 gene lineage, that of specifying petals, in the core eudicots. Whether this function is the result of true neofunctionalization due to gain-of-function mutations has not been demonstrated because both TM6 and euAP3 genes in tomato and petunia were able to carry out a function in petal specification (e.g., ectopic 35S-driven TM6 expression could rescue the euap3 mutant petal phenotype in both species).

The results obtained thus far might be explained solely by subfunctionalization due to differential loss-of-function mutations, both in upstream regulatory regions (causing differential tissue-specific expression) and within the coding sequence (causing differential activities in protein–protein interactions). These studies thus provide an excellent example of subfunctionalization in a homeotic gene lineage and offer an attractive explanation for how such key regulatory gene functions can diversify. Furthermore, such subfunctionalization appears to be quite plastic, in that petunia and tomato, despite having both euAP3 and TM6 genes, have parsed these functions in different ways. Further investigations into the biochemical activity of these proteins in the Solanaceae and in other species that carry only one of these two gene lineages (euAP3 or TM6) will help to answer questions of neofunctionalization and may aid the discovery of the origins of petal specification in the angiosperms.

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Related articles in Plant Cell:

Analysis of the Petunia TM6 MADS Box Gene Reveals Functional Divergence within the DEF/AP3 Lineage

Anneke S. Rijpkema, Stefan Royaert, Jan Zethof, Gerard van der Weerden, Tom Gerats, and Michiel Vandenbussche
Plant Cell 2006 18: 1819-1832. [Abstract] [Full Text]  

Functional Analyses of Two Tomato APETALA3 Genes Demonstrate Diversification in Their Roles in Regulating Floral Development

Gemma de Martino, Irvin Pan, Eyal Emmanuel, Avraham Levy, and Vivian F. Irish
Plant Cell 2006 18: 1833-1845. [Abstract] [Full Text]  

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