A striking aspect of ovule development is a strong genetic overlap with other aspects of plant development. In some cases these overlaps may indicate serial homology, while in other cases they may result from a genetic predisposition to utilize existing pathways during development in plants (i.e., process homology). For example, recent studies have shown that adaxial–abaxial polarity mechanisms are required for integument formation (Kelley et al. 2009; McAbee et al. 2006; Villanueva et al. 1999) in a similar fashion to what has been observed in leaves (Eshed et al. 2001; Kerstetter et al. 2001; McConnell and Barton 1998; McConnell et al. 2001). These results also revealed differences in how these pathways are utilized between integument and leaf development, consistent with integuments having a separate derivation or having significantly diverged during specialization.

Integuments and leaves also exhibit similarity at the molecular level as many of the same genes (or members of gene families) display similar patterning and/or activities (Fig. 1). This sharing of genetic programs between ovules and vegetative organs demonstrates that molecular pathways in plants can be both modular and flexible with respect to their spatio-temporal expression patterns and their mode of action. There are numerous examples that illustrate this concept; we will highlight a number of the key pathways below.

The first example involves genes required for meristem maintenance, namely WUSCHEL (WUS) and SHOOT MERISTEMLESS (STM), which are also active during ovule development (Gross-Hardt et al. 2002; Long et al. 1996). While STM is expressed in the placental (meristematic) region of the carpel during ovule initiation, WUS is expressed in the nucellus early in ovule development and is required for integument formation. Other genes involved in cell proliferation, such as members of the ERECTA kinase family, also play roles in both leaf and integument formation (Pillitteri et al. 2007).

One gene that appears to be a central contributor to integument and primary lateral organ formation and growth is AINTEGUMENTA (ANT), encoding an AP2 family transcription factor. ANT is expressed in lateral organ primordia such as leaves and petals, as well as in the chalaza early during ovule development, marking the site of integument formation (Elliott et al. 1996). Complete loss of ANT function leads to sterile ovules without integuments and reduction in the size and number of primary lateral floral organs (Baker et al. 1997; Elliott et al. 1996; Klucher et al. 1996). In leaves, loss of ANT activity in combination with mutations in YABBY genes such as FILAMENTOUS FLOWER results in polarity defects (Nole-Wilson and Krizek 2006). Therefore, in addition to its role in integument formation, ANT is also a positive regulator of genes specifying either adaxial or abaxial fates in leaves such as PHABULOSA (PHB) (Nole-Wilson and Krizek 2006). Over/ectopic expression of ANT under control of a viral promoter leads to an increase in the size of leaves and primary lateral floral organs further highlighting the role of this transcription factor in organ growth (Krizek 1999; Mizukami and Fischer 2000). Thus, the role of ANT in primary lateral organs and integuments is similar, but it has a fundamental role in initiation and growth of integuments, and a lesser role in these same processes in primary lateral organs.

Another set of genes that are major determinants of integument and leaf formation are those involved in polarity establishment. These genes function through three main pathways, including (i) Class III Homeodomain-leucine zipper (HD-ZipIII) genes and their interactions with KANADI (KAN) genes and microRNAs 165 and 166 (Rhoades et al. 2002) (Emery et al. 2003); (ii) YABBY genes and their interactions with ad/abaxial polarity pathways and class 1 KNOX genes and (Kumaran et al. 2002); (iii) ETTIN-AUXIN RESPONSE FACTOR4 acting with KAN genes to promote abaxial cell identity (Fig. 1; Pekker et al. 2005). Many of these same pathways are utilized during ovule development; we will first highlight the similarities and then discuss the differences.

In integuments, the HD-ZipIII-KAN pathway is active in both integuments, and one YABBY family member, INNER NO OUTER (INO), positively contributes to outer integument formation (Fig. 1; Kelley et al. 2009; Villanueva et al. 1999). The HD-ZIPIII genes PHB, PHAVOLUTA (PHV), and CORONA (CNA) are expressed adaxially in both leaf (Fig. 1a) and inner integument (Fig. 1b) primordia (Kelley et al. 2009; Sieber et al. 2004a). MicroRNA resistant forms of HD-ZIPIII genes produce effects on both leaf (McConnell et al. 2001; Rhoades et al. 2002) and integument (Kelley et al. 2009; Sieber et al. 2004b) development, indicating that this mode of regulation may also be conserved. ABERRANT TESTA SHAPE/KANADI4 (ATS/KAN4) is expressed in the abaxial domain of the inner integument (McAbee et al. 2006) while INO and KAN1, 2 are expressed in the abaxial region of the outer integument (Kelley et al. 2009; Villanueva et al. 1999 and data not shown). Loss of function of these genes, either alone or in combination, results in amorphous or arrested integument growth (Kelley et al. 2009; McAbee et al. 2006). All of these genes therefore act together to positively regulate integument formation as they do in leaves to promote blade growth. Whether or not ANT also acts in these pathways during integument formation is not known, but it is an intriguing possibly given that ANT expression precedes INO, ATS, and HD-ZIPIII gene expression during ovule development.

In comparison, the origin and homology of the outer integument is less clear (Doyle 2006, 2008). Given that unitegmic gymnosperms are the sister group to flowering plants, we can assume that the outer integument was gained somewhere along the stem lineage to angiosperms. With respect to homology, current hypotheses support transformation from a leaf-like organ called a cupule, which was present in such groups as glossopterids and Caytonia, and are hypothesized to be sister to angiosperms (Doyle 2006, 2008). Molecular developmental data can help address questions regarding the origin of integuments. For example, if the outer integument was derived from a cupule (which is a leaf-like structure) then this structure would be expected to share gene expression patterns with a leaf of the same organism because they would be serial homologs. In other words, if there were subsequent diversification of a homologous identity program, then homologous genes should be expressed in homologous structures. In the case of leaf-like organs this would include HD-ZIPIII, KANADI, and YABBY genes. Current genetic analyses (as described above and diagramed in Fig. 1) are congruent with these ideas and provide further support for classical morphological hypotheses regarding the origin of seed plant integuments (Gasser et al. 1998). Considering that all primary lateral organs appear to be serially homologous (Jaramillo and Kramer 2007b) conservation of genetic pathways for creating a laminar organ in both cases is not surprising.

As previously mentioned, genetic and molecular analyses in Arabidopsis have defined key regulatory networks in ovule development. Comparisons with other species are needed to validate these networks on an evolutionary scale to determine the degree of conservation or divergence these programs exhibit. Knowledge of ovule development in seed plants beyond Arabidopsis is also necessary to further our general understanding of genetic control of ovule development. This should include (i) gymnosperm ovules, which are unitegmic (i.e., possess only one integument) and orthotropous (upright), (ii) basal angiosperm groups such as ANITA grade members and magnoliids, and (iii) derived angiosperm crown group members, most notably the asterids, which are predominantly unitegmic.

Whereas most gymnosperm taxa are not genetically tractable, there are opportunities for comparative studies between angiosperm groups that represent meaningful phylogenetic positions. For instance, studies on INO function and expression are related to ideas regarding (i) the outer integument as an angiosperm synapomorphy and (ii) the origin of unitegmic ovules in angiosperms. An example is provided by one such comparative study that examined the differences between Arabidopsis and a basal asterid genus Impatiens (McAbee et al. 2005). Although integument number is reduced from two to one in most asterids, including tomato and petunia, it is somewhat plastic within the genus Impatiens where there are species with bitegmic, intermediate, and unitegmic ovules (McAbee et al. 2005). This morphological transition could have occurred through loss of either integument or through fusion of the two integuments into one. Through histological analysis and expression analyses of INO in Impatiens species with varied integument character McAbee et al. (2005) were able to distinguish between these two hypotheses and show that in Impatiens the bitegmic to unitegmic transition is the result of congenital fusion of the two integuments. Notably, the pattern of expression of INO orthologs was conserved in bitegmic and unitegmic species, indicating a likely conservation of INO gene function between rosids and asterids.

Another approach requires the presence of phylomimicking mutants in a genetically tractable taxon. These are mutants that phenocopy the morphology of a naturally occurring taxon (Mummenhoff et al. 2009). There are three such Arabidopsis mutants that display integument defects that are morphologically similar to those in derived angiosperm clades. One is inner no outer (ino), which lacks an outer integument (Villanueva et al. 1999). While the transition to unitegmic ovules in Impatiens appears to have occurred via fusion, unitegmic ovules may have arisen by loss of the outer integument in other groups such as Piperaceae (Bouman 1984). In such cases direct examination of INO gene structure and expression patterns could provide new insight into how these unitegmic ovules arose. Another excellent candidate phylomimicking mutant is ats, in which the two Arabidopsis integuments have been congenitally fused into a single structure (McAbee et al. 2006). A comparison of ATS expression patterns and sequences among Impatiens species could be informative, if loss of ATS activity were to be causative with respect to the fusion phenotype in this genus. A final example is seen in the ant mutant (Elliott et al. 1996; Klucher et al. 1996). The lack of integuments in ant ovules is reminiscent of ategmic ovules reported in some members of the order Santalales (Bouman 1984). In this case, does loss of ANT function correlate with the naturally occurring phenotypic change or is there some other genetic mechanism that is responsible? Expression studies of ANT orthologs between Santalales taxa with bitegmic and ategmic ovules could help provide support toward either hypothesis.

Examination of an INO ortholog from Nymphaea, a basal angiosperm, showed that NaINO is expressed strongly in the outer integument as well as the inner integument and nucellus, supporting the hypothesis that INO may have a conserved role in the outer integument among angiosperms (Yamada et al. 2003). Further study on INO orthologs in other early divergent angiosperm taxa with bitegmic ovules is required to determine whether the expanded NaINO expression pattern is unique to Nymphaea or is representative of the ancestral INO expression pattern. Consistent with a role only in outer integument development, current phylogenetic analyses indicate that INO appears to be angiosperm specific (Floyd and Bowman 2007; Lee et al. 2005) and would therefore be representative of a neo-functionalization among YABBY genes.