Fruit evolution

Fruits are novel structures resulting from transformations in the late ontogeny of the carpels that evolved in the flowering plants (Doyle, 2013). Fruits are generally formed from the ovary wall but accessory fruits (e.g., apple and strawberry) may contain other parts of the flower including the receptacle, bracts, sepals, and/or petals (Esau, 1967; Weberling, 1989). For purposes of comparison we will discuss fruits that develop from the carpel wall only. Fruit development generally begins after fertilization when the carpel wall (pericarp) transitions from an ovule containing, often photosynthetic vessel, to a seed containing dispersal unit. The fruit wall will differentiate into endocarp (1-few layers closest to developing seeds, often inner to the vascular bundle), mesocarp (multiple middle layers, including the vascular bundles and outer tissues), and exocarp (for the most part restricted to the outermost layer, and only occasionally including hypodermal tissues) (Richard, 1819; Sachs, 1874; Bordzilowski, 1888; Farmer, 1889; Roth, 1977; Pabón-Mora and Litt, 2011). Fruits are classified by their number of carpels, whether multiple carpels are free or fused, texture (dry or fleshy), how the pericarp layers differentiate and whether and how the fruits open to disperse the seeds contained inside (Roth, 1977).

There is a vast amount of fruit morphological diversity and fruit terminology that corresponds to this diversity (reviewed in Esau, 1967; Weberling, 1989; Figure ​Figure1).1). For example, fruits made of a single carpel include follicles or pods (e.g., Medicago truncatula; Figure ​Figure1D)1D) and sometimes drupes (e.g., Ascarina rubricaulis; Figure ​Figure1K).1K). Follicles and pods both have thick walled exocarp and thin walled parenchyma cells in the mesocarp. However, follicles also have thin walled parenchyma cells in the endocarp while many pods have a heavily sclerified endocarp with 2 distinct layers with microfibrils oriented in different directions (Roth, 1977). When follicles mature the parenchyma and schlerenchyma cell layers dry at different rates causing the fruit to open at the carpel margins (adaxial suture) while pods open at the carpel margin and the median bundle of the carpel due to additional tensions in the endocarp (Roth, 1977; Fourquin et al., 2013). Fruits that are multicarpellate but not fused can include follicles that are free on a receptacle (e.g., Aquilegia coerulea; Figure ​Figure1H).1H). Fruits that are multi-carpellate and fused include berries (e.g., Solanum lycopersicum, Carica papaya, and Vitis vinifera; Figures 1B,C,E), capsules (e.g., Arabidopsis thaliana, Eschscholzia californica, Papaver somniferum; Figures 1A,F,G), caryopses (grains of Oryza sativa and Zea mays; Figures 1I,J), and drupes (e.g., peach). These multicarpellate fruits differ by the differentiation of the pericarp and their dehiscence mechanisms. Berries and drupes tend to be indehiscent and the pericarp of berries is often fleshy and composed mainly of parenchyma tissue (Richard, 1819; Roth, 1977). The endocarp and mesocarp of drupes is also fleshy, however, the endocarp is composed of highly sclerified tissue termed the stone (Richard, 1819; Sachs, 1874). Caryopses are also indehiscent and have a thin wall of pericarp fused to a single seed (Roth, 1977). Capsules can have few to many cells in the pericarp and the different layers of the pericarp can be composed of parenchyma tissue in most layers and sclerenchyma tissue in the mesocarp and/or endocarp. Capsules can dehisce at various locations including at the carpel margins (septicidal), at the median bundles (loculicidal) or through small openings (poricidal) (Roth, 1977). The extreme fruit morphologies found across angiosperms, even in closely related taxa suggest that fruits are an adaptive trait, thus, homoplasious seed dispersal forms and transformations from berries to capsules or drupes and vice versa are common in many plant families (Pabón-Mora and Litt, 2011).

The molecular basis that underlies fruit diversity is not well-understood. However, the fruit molecular genetic network in Arabidopsis thaliana (Arabidopsis), necessary to specify the different components of the fruit including the sclerified (lignified) tissues necessary for the controlled opening (dehiscence) of the fruit are well-characterized (Reviewed in Ferrándiz, 2002; Roeder and Yanofsky, 2006; Seymour et al., 2013). Arabidopsis fruits develop from two fused carpels and are specialized capsules called siliques, which open along a well-defined dehiscence zone (Hall et al., 2002: Avino et al., 2012). The siliques are composed of two valves separated by a unique tissue termed the replum present only in the Brassicaceae. The valves develop from the carpel wall and are composed of an endocarp, mesocarp and exocarp. The replum and valves are joined together by the valve margin. The valve margin is composed of a separation layer closest to the replum and liginified tissue closer to the valve. The endocarp of the valves becomes lignified late in development and plays a role, along with the lignified layer and separation layer of the valve margin, in fruit dehiscence (Ferrándiz, 2002).

Developmental genetic studies in Arabidopsis thaliana have uncovered the genetic network that patterns the Arabidopsis fruit. FRUITFULL (FUL) is necessary for proper valve development and represses SHATTERPROOF 1/2 (SHP 1/2) (Gu et al., 1998; Ferrándiz et al., 2000a). SHP1/2 are necessary for valve margin development (Liljegren et al., 2000). REPLUMLESS (RPL) is necessary for replum development and represses SHP1/2 (Roeder et al., 2003). The repression of SHP1/2 by FUL and RPL keeps valve margin identity to a small strip of cells. SHP1/2 activate INDEHISCENT (IND) and ALCATRAZ (ALC), which are both necessary for the differentiation of the dehiscence zone between the valves and replum (Girin et al., 2011; Groszmann et al., 2011). IND is important for lignification of cells in the dehiscence zone while IND and ALC are necessary for proper differentiation of the separation layer (Rajani and Sundaresan, 2001; Liljegren et al., 2004: Arnaud et al., 2010). SPATULA (SPT) also plays a minor role, redundantly with its paralog ALC in the specification of the fruit dehiscence zone (Alvarez and Smyth, 1999; Heisler et al., 2001; Girin et al., 2010, 2011; Groszmann et al., 2011).

FUL, SHP1/2, RPL, IND, SPT, and ALC all belong to large transcription factor families. FUL and SHP1/2 belong to the MADS-box family (Gu et al., 1998; Liljegren et al., 2000), IND, SPT, and ALC belong to the bHLH family and RPL belongs to the homeodomain family (Heisler et al., 2001; Rajani and Sundaresan, 2001; Roeder et al., 2003; Liljegren et al., 2004). Some of these transcription factors are known to be the result of Brassicaceae specific duplications, others seem to be the result of duplications coinciding with the origin of the core eudicots (Jiao et al., 2011). For instance SHP1 and SHP2 are AGAMOUS paralogs and Brassicaceae-specific duplicates belonging to the C-class gene lineage (Kramer et al., 2004). FUL is a member of the AP1/FUL gene lineage unique to angiosperms (Purugganan et al., 1995). FUL belongs to the euFULI clade, that together with euFULII and euAP1 are core-eudicot specific paralogous clades. Nevertheless, pre-duplication proteins are similar to euFUL proteins, hence they have been named FUL-like proteins and are present in all other angiosperms (Litt and Irish, 2003). Likewise, ALC and SPT and IND are the result of several duplications in different groups of the bHLH family of transcription factors, but the exact duplication points have not yet been identified (Reymond et al., 2012; Kay et al., 2013). Hence, it is unclear whether this gene regulatory network can be extrapolated to fruits outside of the Brassicaceae. Functional evidence from Anthirrhinum (Plantaginaceae) (Müller et al., 2001), Solanum(Solanaceae) (Bemer et al., 2012; Fujisawa et al., 2014), and Vaccinium (Ericaceae) (Jaakola et al., 2010) in the core eudicots, as well as Papaver and Eschscholzia (Papaveraceae, basal eudicots) (Pabón-Mora et al., 2012, 2013b) suggest that at least FUL orthologs have a conserved role in regulating proper fruit development even in fruits with diverse morphologies. euFUL and FUL-like genes control proper pericarp cell division and elongation, endocarp identity, and promote proper distribution of bundles and lignified patches after fertilization. However, functional orthologs of SHP, IND, ALC, SPT, or RPL have been less studied and it is unclear whether they are conserved in core and non-core eudicots. The limited functional data gathered suggests that at least in other core eudicots SHP orthologs play roles in capsule dehiscence (Fourquin and Ferrandiz, 2012) and berry ripening (Vrebalov et al., 2009). Likewise, SPT orthologs have been identified as potential key players during pit formation in drupes, likely regulating proper endocarp margin development (Tani et al., 2011). RPL orthologs have not been characterized in core eudicots, but an RPL homolog in rice is a domestication gene involved in the non-shattering phenotype, suggesting that the same genes are important to shape seed dispersal structures in widely divergent species (Arnaud et al., 2011; Meyer and Purugganan, 2013). At this point, more expression and functional data are urgently needed to test whether the network is functionally conserved across angiosperms, nevertheless, all these transcription factors are candidate regulators of proper fruit wall growth, endocarp and dehiscence zone identity, and carpel margin identity and fusion (Kourmpetli and Drea, 2014). In the meantime, another approach to study the putative conservation of the network is to identify how these specific gene families have evolved in flowering plants as duplication and diversification of transcription factors are thought to be important for morphological evolution. Although, based on gene analyses no functions can be explicitly identified, the presence and copy number of these genes will provide testable hypothesis for future studies in different angiosperm groups. Thus, to better understand the diversity of fruits and the changes in the fruit core genetic regulatory network we analyzed the evolution of these transcription factor families from across the angiosperms. We utilized data in publicly available databases and performed phylogenetic analyses. We found different patterns of duplication across the different transcription factor families and discuss the results in the context of the evolution of a developmental network across flowering plants.