The fruit, the whole fruit, and everything about the fruit.

Journal of Experimental Botany, Vol. 65, No. 16, pp. 4491–4503, 2014 doi:10.1093/jxb/eru144 Advance Access publication 10 April, 2014

Review paper

The fruit, the whole fruit, and everything about the fruit Sofia Kourmpetli1 and Sinéad Drea1,* 1

Department of Biology, University of Leicester, University Road, Leicester LE1 7RH, UK

* To whom correspondence should be addressed. E-mail: [email protected] Received 14 January 2014; Revised 14 January 2014; Accepted 5 March 2014

Fruits come in an impressive array of shapes, sizes, and consistencies, and also display a huge diversity in biochemical/metabolite profiles, wherein lies their value as rich sources of food, nutrition, and pharmaceuticals. This is in addition to their fundamental function in supporting and dispersing the developing and mature seeds for the next generation. Understanding developmental processes such as fruit development and ripening, particularly at the genetic level, was once largely restricted to model and crop systems for practical and commercial reasons, but with the expansion of developmental genetic and evo-devo tools/analyses we can now investigate and compare aspects of fruit development in species spanning the angiosperms. We can superimpose recent genetic discoveries onto the detailed characterization of fruit development and ripening conducted with primary considerations such as yield and harvesting efficiency in mind, as well as on the detailed description of taxonomically relevant characters. Based on our own experience we focus on two very morphologically distinct and evolutionary distant fruits: the capsule of opium poppy, and the grain or caryopsis of cereals. Both are of massive economic value, but because of very different constituents; alkaloids of varied pharmaceutical value derived from secondary metabolism in opium poppy capsules, and calorific energy fuel derived from primary metabolism in cereal grains. Through comparative analyses in these and other fruit types, interesting patterns of regulatory gene function diversification and conservation are beginning to emerge. Key words: Capsule, cereals, fruit, grain, metabolism, poppy.

Introduction The fruit: from initiation to utilization The ability to conduct research on myriad aspects of fruit development from organ initiation through development to usable end product (Fig.1) in phylogenetically and morphologically diverse species that have agricultural, medicinal, and ecological value provides an opportunity to comprehensively understand the fruit and its origin in the plant kingdom. The story of the fruit poses intriguing questions for every biological discipline to address, as well as posing questions at multiple levels of analyses (Fig. 1). As a notable botanical innovation in the arrival of the angiosperms, to being the primary human food source, the characterization of each chapter in the story of the fruit requires taxonomists, developmental biologists, bioinfomaticians, biochemists, and botanists. The links between the different levels of

investigation, particularly taxonomy, phylogeny, and development are becoming stronger in the area of comparative development or evolution of development. Biologists have always been interested in the evolution of developmental processes, but advances in molecular techniques now complement earlier analyses, which were inevitably descriptive and based on gross morphological characteristics. The ability to extract and sequence DNA from diverse species is still the staple of phylogenetic analyses, but advances in sequencing technology and adaptation of other molecular technologies has taken phylogenetic analysis into a whole new era to the point that the concept of ‘model organism’ is becoming commonplace, at least to some degree depending on the definition of what constitutes a ‘model’ (Abzhanov et al., 2008; Rowan et al., 2011). There is perhaps still a

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Abstract

4492 | Kourmpetli and Drea

distance between developmental studies focusing on early stages in fruit development and the metabolic/biochemical analysis of the end product. However, at some point downstream in the fruit’s genetic regulatory network (GRN), the master regulators at the top of the hierarchy that control organ identity, specification, and development of specialized fruit tissues (Table 1) must link to the ‘local’ regulators of specific metabolic and biochemical pathway components. In the case of AGAMOUS (AG), the MADS-box gene specifying carpel identity in Arabidopsis, a recent systematic identification of its targets using extensive transcriptomic data highlighted that a large proportion of direct targets were also transcription factors (TFs), including CRABS CLAW (CRC), the YABBY gene also important in fruit development (O’Maoileidigh et al., 2013). Diversification in regulatory gene expression and/or function anywhere in this chain undoubtedly accounts for the massive variation in plants generally and fruits specifically (Carroll, 2008). Comparative analyses across the angiosperms using key regulator genes (Table 1) as candidates is revealing intriguing patterns of conservation and diversification in function high up in this regulatory hierarchy. In this review we focus on two cultivated fruit types we have been involved with directly, the poppy capsule and the cereal grain. Fig. 2 puts these case studies in the context of the angiosperm phylogeny, and in this review we summarize research that encompasses basic development and its applied relevance (Fig. 1). Our focus is on the TFs controlling these diverse processes from initiation to end-use. As traits selected for through agriculture involve fundamental plant developmental processes, it is not surprising that TFs are being identified as the determinants of these processes and the bases of the genetic differences distinguishing wild and cultivated species (Doebley et al., 2006; Tables 1 and 3).

Most of the work published on the genetic basis of fruit development in eudicot species has been focused on core eudicots, such as the model Arabidopsis, crop tomato, and ornamental Petunia (Table 1). However, given the wide diversity of fruit forms and the practical/economic significance of key fruit traits in commercially important species (abscission, dehiscence, ripening duration, longevity, seed production), a more detailed study of fruit development in other angiosperm groups, such as basal eudicot species, is extremely interesting from an evolutionary point of view. The order Ranunculales represents the earliest diverging eudicot order and includes species that show a great variety of flower and fruit forms. Within this order, the Papaveraceae and Ranunculaceae families are the focus of recent research (Table 2). In addition, in terms of cereal research, Brachypodium distachyon has been emerging in the last few years as a valuable model species and a taxonomically relevant reference point for the ‘core pooids’, such as wheat, barley, and oat. With its genome already sequenced and several genetic resources already available (IBI, 2010; Mur et al., 2011), a better understanding of its fruit development at a molecular genetics level could help shed light on more complex genomes, such as wheat, and ultimately assist in the genetic improvement of temperate grasses.

Case study: the capsule of Papaver somniferum (opium poppy), a basal eudicot Within the Ranunculales, there are very diverse fruit forms (Table 2). The capsule of opium poppy contrasts to the explosive silique-like fruit of California poppy (Becker et al., 2005). Each species (Table 2) has features that are both shared and distinct from the other, and from the established model species in the core eudicot and monocot clades, such as Arabidopsis,


Fig. 1. Flowchart illustrating the many levels at which research on fruits can be conducted depending on the focus of individual groups, from initiation to point of harvesting and processing. Some specific developmental processes, metabolic products and cell/tissue types are included. (This figure is available in colour at JXB online.)

Fruit development in poppy and cereals | 4493 Table 1. A summary of some of the key fruit developmental genes identified in core eudicots and being examined in species across the angiosperms The list is by no means complete but includes genes that cover a range of constituent tissues related to fruit development and where we aim to highlight recent findings. Gene name

Genus

TF family

Function/ tissue

Reference

Carpel/ fruit

AG TAG1 pMADS3 FAR SHP1/2 FBP6 PLE TAGL1 FUL FUL1 (TDR4) FUL2(MBP7) RIN AGL63/GOA RPL IND ALC AP2 AP2a CRC FAS FBP7/11 STK INO TT16/ABS

Arabidopsis Tomato Petunia Antirrhinum Arabidopsis Petunia Antirrhinum Tomato Arabidopsis Tomato Tomato Tomato Arabidopsis Arabidopsis Arabidopsis Arabidopsis Arabidopsis Tomato Arabidopsis Tomato Petunia Arabidopsis Arabidopsis Arabidopsis

MADS-box MADS-box MADS-box MADS-box MADS-box MADS-box MADS-box MADS-box MADS-box MADS-box MADS-box MADS-box MADS-box Homeobox HLH HLH AP2/EREBP AP2/EREBP YABBY YABBY MADS-box MADS-box YABBY MADS-box

Identity, determinacy Identity, determinacy Development Development Valve margin Development Identity Ripening/composition Valve shattering Ripening Ripening Ripening/composition Fruit cell size Replum - shattering Dehiscence Dehiscence Replum Ripening/composition Polarity, determinacy Locule number (size) Identity/determinacy Seed dispersal Integument Integument

Yanofsky et al., 1990 Pnueli et al., 1994 Tsuchimoto et al., 1993 Davies et al., 1999 Liljegren et al., 2000 Heijmans et al., 2012 Bradley et al., 1993 Pan et al., 2010; Vrebalov et al., 2009 Gu et al., 1998 Bemer et al., 2012 Bemer et al., 2012 Vrebalov et al., 2002 Prasad et al., 2010 Roeder et al., 2003 Liljegren et al., 2004 Rajani and Sundaresan, 2001 Ripoll et al., 2011 Chung et al., 2010; Karlova et al., 2011 Bowman and Smyth, 1999 Cong et al., 2008 Colombo et al., 1995; Heijmans et al., 2012 Pinyopich et al., 2003 Villanueva et al., 1999 Nesi et al., 2002

Ovule/ seed

tomato, and rice, and they therefore offer much material for morphological comparisons to models and crop species. The ovary of Papaver somniferum is globular and supported on a gynophore and receptacle subtended by the pedicel (Fig. 3). It is overlaid by a flat persistent stigmatic surface composed of rays that are fused at the edges and separated by deep invaginations producing stigmatic lobes. Profuse papillae that capture the dehisced pollen grains are found at the ray edges. There is no style. The capsule is designated as poricidal in terms of seed dispersal, the pores forming just beneath the stigmatic rays; however, the capsules can be dehiscent or indehiscent as not all cultivars produce pores. The gynoecium is multicarpellate paracarpous and the edges of the fused carpels form adaxial septae that do not extend across the fruit centre producing a unilocular capsule. The number of stigmatic rays equals the number of carpels. The capsule is open valvate along placentae with distinct valves visible. Numerous ovules develop on the placentae covering the surface of the septae and seeds vary in colour from white to black (Bernath, 1998; Grey-Wilson, 2000; Kapoor, 1995). One of the main drivers in developing functional tools in basal eudicot species is to explore the genetic basis of the development of the varied flower morphologies, and to ascertain at the functional level the degree of conservation or divergence in the genetic mechanisms directing the initiation, specification, and development of the flower and

its composite organs. Clues to these were already obtained using detailed spatial and temporal gene expression analyses in addition to phylogenetics and taxonomy (e.g. Kramer and Irish, 1999; Drea et al., 2007). Within the basal eudicots there are published reports of at least six species being amenable to explicit functional gene studies using VIGS (virus-induced gene silencing; Di Stilio et al., 2010; Goncalves et al., 2013; Gould and Kramer, 2007; Hidalgo et al., 2012; Hileman et al., 2005; Table 2), in addition to the use of mutagenized lines and natural variants (Galimba et al., 2012; Lange et al., 2013), and all have some genomic resources in place and/or in development (Becker et al., 2005; Di Stilio et al., 2005; Kramer, 2009). When considering specific genes through which to compare genetic mechanisms of flower and fruit development in basal eudicots, it is not surprising that members of the ABC(DE) model of flower development derived from early work in Arabidopsis, Antirrhinum, and Petunia (Coen and Meyerowitz, 1991; Heijmans et al., 2012; Ferrario et al., 2004) were prime candidates (Table 2). In relation to fruit development we focus on the studies involving C/D-class analyses in addition to genes from other families because of their expected role in fruit development (Table 1). The MADS-box genes involved in fruit development pose interesting schemes in terms of phylogenetics as well as in terms of the very different fruit forms and their developmental programmes. For example, the SHATTERPROOF


Location


(SHP) C-class and APETALA 1 (AP1) A-class sub-lineages are only found in core eudicots where they control fruit patterning and outer whorl identity in Arabidopsis, respectively (Dreni and Kater, 2014; Kramer et al., 2004, Litt and Irish, 2003). AG orthologues have been studied at the functional level in P. somniferum, Eschscholzia californica, and Thalictrum thalictroides. Analyses of AG genes in these species showed the depth of C-functional conservation (Galimba et al., 2012; Hands et al., 2011; Yellina et al., 2010). In Eschscholzia and Thalictrum duplication has generated two AG paralogues that in Eschscholzia have similar expression profiles (Zahn et al., 2006) and functional redundancy, whereas in Thalictrum one of the paralogues, ThAG2, is expressed in ovules only (Di Stilio et al., 2005). The other ThAG1 paralogue is required for AG floral identity and determinacy functions (Galimba et al., 2012). In opium poppy, alternative splicing at the AG locus generates two transcripts with similar expression and a requirement for both transcripts to mediate the identity and determinacy functions (Hands et al., 2011). Orashakova et al. (2009) studied the role of CRC in detail in Eschscholzia, showing that the gene played a role not only in fruit development and determinacy as in Arabidopsis, but also in ovule development. The FUL genes have been characterized functionally in both P. somniferum and E. californica, where it was shown that the duplicated FUL genes retained the collective roles of the paralogous euAP1 and euFUL in meristem, petal identity, and fruit (where they mediate differentiation of the valve; Gu et al., 1998). Based on these results subfunctionalization was proposed as the consequence of the duplication in the core eudicots (Pabon-Mora et al., 2012; Table 2).

Intriguingly, the FUL genes in Aquilegia showed no role in fruit development and the authors suggested that the floral and reproductive functions were decoupled in this species (Pabon-Mora et al., 2013).

The P. somniferum (opium poppy) capsule as a source of alkaloids As a group of plants the Ranunculales are also distinctive in that they are the main producers of the BIA (benzylisoquinilone alkaloid) compounds with E. californica, P. somniferum, and Thalictrum (flavum and tuberosum) being some of the most intensively studied species in this regard (Ziegler and Facchini, 2008). BIAs are specialized metabolites that are often associated with powerful pharmacological effects including analgesic, antibacterial, relaxant, cough suppressant, and anticancer properties (Hagel and Facchini, 2013). Opium poppy is distinctive in its production of the valuable morphinan alkaloids including thebaine, codeine, and morphine, which are the main components of its latex. The latex is essentially the cytoplasm of specialized cells called laticifers; specific to certain families throughout the angiosperms and classified based on their development and morphology (Hagel et al., 2008). Esau (1953) classified the laticifers of Papaver as being articulated anastomosing—chains of cells (forming vessels), where the end walls are perforated and further connected to other vessels. They are located adjacent to, and develop simultaneously with, the phloem tissue (Hagel et al., 2008) and their path is followed by tracking the course of the vascular bundle. The opium poppy’s capsular fruit is synonymous with alkaloid production (Fig. 3). Alkaloid biosynthesis is not


Fig. 2. Simplified land plant phylogeny (based on figure in Rowan et al., 2011) to indicate basic relationships of various genera and species discussed.

Fruit development in poppy and cereals | 4495 Table 2. A summary of recent basal eudicot research highlighting functional analyses of flower genes in Ranunculales Family

Genus

Fruit features

Flower genes studied

Reference

Papaveraceae

Eschscholzia

Bivalved, explosive

FLO, MADS-box (AP3, PI, AG, FUL), YABBY (CRC)

Papaver

Poricidal capsule

MADS-box (AP3, PI, AG, FUL), YABBY (FIL)

Cyscicapnos Thalictrum

Bicarpellate, balloon-like Open, uniovule, ascidiate

FLO MADS-box (AG)

Aquilegia

apocarpic

MADS-box (AP3, PI, FUL)

Nigella

Inflated, capsule; syncarpous

MADS-box (AP3)

Orashakova et al., 2009; Pabon-Mora et al., 2012; Wege et al., 2007; Yellina et al., 2010 Pabon-Mora et al., 2012; Drea et al., 2007; Hands et al., 2011; Vosnakis et al., 2012 Hidalgo et al., 2012 Di Stilio et al., 2010; Galimba et al., 2012 Kramer et al., 2007; Pabon-Mora et al., 2013 Goncalves et al., 2013

Ranunculaceae

Location

Gene name

Genus

TF Family

Function

Reference

Arabidopsis orthologue

Surrounding tissues

NUD TGA1 qSH1 SH4* AP2(SHAT1) SH1 Q VRS1 HvAP2 DL OsMADS3/58

Hordeum Zea Oryza Oryza Oryza Sorghum Triticum Hordeum Hordeum Oryza Oryza

AP2/EREBP SBP-binding homeobox Myb-like AP2/EREBP YABBY AP2/EREBP HD-ZIP AP2/EREBP YABBY MADS-box

Adhering hull (palea/lemma) Seed casing (cupule) Grain shattering Grain shattering Grain shattering Grain shattering Grain size Two-row to six-row barley Grain density Carpel identity Carpel development

WIN1/SHN1

OsMADS13/21 GT1

Oryza Zea

MADS-box HD-ZIP

Ovule identity Carpel development and number

RC RSR1 OSMADS29 ZmMRP-1 OPAQUE2

Oryza Oryza Oryza Zea Zea

HLH AP2/EREBP MADS-box Myb bZIP

BLZ1/2 HvMYBS3 GAMYB BPBF SAD MCB1

Hordeum Hordeum Hordeum Hordeum Hordeum Hordeum

bZIP Myb Myb DOF DOF Myb

Grain colour (pericarp) Starch Nucellus degradation Transfer cell differentiation Storage protein gene activation Storage protein/gene activation GA response/germination GA response GA response GA response GA response

Taketa et al., 2008 Wang et al., 2005 Konishi et al., 2006 Li et al., 2006 Zhou et al., 2012 Lin et al., 2012 Simons et al., 2006 Komatsuda et al., 2007 Houston et al., 2013 Yamaguchi et al., 2004 Yamaguchi et al., 2006; Dreni et al., 2011 Dreni et al., 2007 Whipple et al., 2011; Wills et al., 2013 Sweeney et al., 2006 Fue and Xue, 2010 Yin and Xue, 2012 Gomez et al., 2002, 2009 Hartings et al., 1989

Shattering from rachis

Grain number/size

Carpel/ovule

Endosperm domains

restricted to the capsule, however, and alkaloids are produced throughout the poppy plant after the laticifers have differentiated in the seedlings. At the base of the capsule the vascular bundles fuse forming several amphicribral (where the xylem is surrounded by phloem) bundles (Kapoor, 1973; Fukuda, 2004), and the organization and concentration of

RPL AP2 YAB2 AtTOE1 ATHB-21 AP2 CRC AG STK

TT8 AP2 AGL32/ABS BZO2H3

Onate et al., 1999 Diaz et al., 2002 Mena et al., 2002 Isabel-LaMoneda et al., 2003 Rubio-Somoza et al., 2006

MYB33/65/101

the vascular tissue and associated laticifers contributes to proficient latex production in the fruit. The seeds are actually free from alkaloids as the laticiferous vessels terminate in the placenta and do not follow through to the developing ovule. The seeds produced by the capsule are also familiar as toppings for bagels and other bread products, and are rich


Table 3. Summary of some key TFs identified in grasses affecting various aspects of grain number, specification, development, composition, and harvesting


in oils that can also be used in producing paints and varnishes (Bernáth, 1998a). Whereas the production of many alkaloids can be induced in cell cultures, this has only been successful for sanguinarine in opium poppy, and not for the morphinan alkaloids (Zulak et al., 2008), and so the cultivation of opium poppy plants is still an essential part of morphine production. This has been suggested to be because the cell cultures do not possess the differentiated laticifers present in seedlings and older plants (Kutchan et al., 1983). When the flower opens the already large capsule continues to increase markedly in size for three weeks with the vertical section area of the capsule increasing more than 3-fold within the first week (Kapoor, 1995). Images of lanced capsules oozing latex are commonly used to illustrate the process of how opium and alkaloids are obtained from the plant, and this still continues to be the means of harvesting in some areas (Bernáth, 1998b). Two weeks after petal fall the capsule has reached its maximum size, but is still green, and it is at this point that the first incision or lancing is performed. The instrument used is a knife with three or four blades about 2 mm apart, and the capsules are scored longitudinally to or from just below the stigmatic rays (Kapoor, 1995). The depth of incision is vital, as if scored too deeply the latex can seep internally, contaminating seeds (CONTAM, 2011), rather than aggregate externally on the surface of the capsule from where it is collected. However, this massively laborious and intricate means of extraction has been superceded by the Kabay method (named after the Hungarian chemist who developed the process) in which the alkaloids are retrieved from poppy ‘straw’ (Németh, 1998) by mechanical harvesting using modified combine harvesters. In this method the poppy capsules and 10–20 cm of the stem are harvested, allowed to dry completely, and seeds separated before processing the remaining material. The knowledge that alkaloids were associated with the latex led to the gradual identification of the many intermediates

and enzymes in the alkaloid biosynthetic pathway. A recent review of BIA biosynthesis (Hagel and Facchini, 2013) listed 25 enzymes in the intricate biosynthetic pathways of the BIAs berberine, morphine, sanguinarine, codeine, and thebaine for which corresponding genes have been identified. This provided the means (probes and antibodies) to map the synthesis sites of the alkaloids leading to the discovery that the sieve elements of the phloem were directly involved (Bird et al., 2003; Weid et al., 2004). This was a significant discovery implicating sieve elements in complex metabolic processes and not just transport of solutes. As well as facilitating the discovery of much about the nature of laticiferous vessels and the proactive role of phloem in plant metabolism, the biosynthesis of alkaloids in poppy capsules also enhanced our understanding of genomic features that underlie the production of natural substances in plants. Comparative transcriptomics using a high-noscapine yielding cultivar identified the biosynthetic enzymes organized as a gene cluster in the genome (Winzer et al., 2012). This study provided another example of the association of the biosynthesis of natural plant products with gene clusters such as avenicin in oat roots (DellaPenna and O’Connor, 2012). It is satisfying that much of this recent progress in characterizing the BIA biosynthesis pathways and its components in opium poppy has been facilitated by the development of tools for evo-devo analyses such as VIGS, though there are caveats and limitations with this technique when studying either BIA biosynthesis or developmental control genes (Hagel and Facchini, 2013; Geuten et al., 2013).

Case study: the grain of temperate cereals The fruit of the grasses is as distinctive botanically as it is valuable economically. In contrast to fleshy-fruited crops such as tomato and the alkaloid-producing capsule of poppy, it is the single seed within the fruit that holds the economically


Fig. 3. Papaver somniferum (opium poppy). Left; photograph of a flower of cultivar Persian white with inset of solitary capsule. Right; schematic showing capsule in longitudinal profile and capsule in transverse profile. A close-up of the vascular bundle and laticifers is shown bottom right. Drawings based on Petri and Mihalik (1998), Kapoor (1995), and Ziegler and Facchini (2008).

Fruit development in poppy and cereals | 4497 was assigned to the CRC orthologue DROOPING LEAF (DL), where mutants produced stamens instead of carpels (Nagasawa et al., 2003; Yamaguchi et al., 2004). More recent studies, however, using a suite of insertion mutants, showed that both AG orthologues redundantly controlled female reproductive organ identity suggesting that AG-control of carpel identity was indeed conserved in eudicots and monocots (Dreni et al., 2011). To date, rice remains the only monocot for which we have explicit functional data on all the C and D class genes (Dreni and Kater, 2014). Maize differs significantly from rice, wheat, and barley in having separate male and female flowers. The male flowers follow a developmental programme that represses growth of a carpel, whereas in the female flowers the carpel develops into characteristic corncobs or ears. The HD-ZIP gene, GT (GRASSY TILLERS1), was identified in a screen for floral development mutants in maize where the growth of carpels in the male flower was derepressed in the gt1 mutant (Whipple et al., 2011). The ability of GT1 to repress lateral growth is manifested in the number of ears produced in the female flowers (Wills et al., 2013) where it was identified as the basis of the prol1.1 (prolificacy) locus where increased GT1 expression in maize compared with ancestral (and multipleear forming) teosinte suppressed the initiation of secondary ear buds (Wills et al., 2013). Grain number increase in barley domestication was associated with a transition from two-row to six-row spikes. This transition was found to be controlled by the VRS1 gene, which encodes a class 1 HD-ZIP TF that acted in wild barley by suppressing the growth or lateral meristems (Komatsuda et al., 2007), and is also paralogue of GT1 (Whipple et al., 2011). A modifier of the 2-to-6 row transition, intermedium-C, was identified and encodes the orthologue of TB1 (TEOSINTE BRANCHED1; Ramsay et al., 2011), the main determinant of apical dominance in cultivated maize (Doebley et al., 1997). Fewer master regulators specific to the development of the grain (pericarp and seed within) have been identified (Sabelli and Larkins, 2009). In cultivated cereals and most grasses, the endosperm is the largest compartment in the harvested grain and is differentiated into functionally distinct subdomains, which can include the aleurone, modified aleurone or transfer layer (at the junction of maternal vasculature and endosperm), central starchy endosperm, and embryo-surrounding region endosperm. The organization of the surrounding maternal fruit and seed tissues varies, affecting the possible routes by which sugars and amino acids are supplied to the developing endosperm. For example, the generally accepted conduit for maternal nutritional supplies in wheat grains is through the nucellar projection and modified aleurone (Wang et al., 1995; Fig. 4). Rice, however, also uses another pathway via the nucellar epidermis (Oparka and Gates, 1981). OsMADS29 was recently shown to control nucellus degeneration in the developing rice grain with reduced expression producing shrunken seeds and reduced starch synthesis (Yin and Xue, 2012). Degeneration of the nucellus, and of the surrounding maternal tissues generally, is an important accompaniment to endosperm and embryo growth in cereal grains. This gene is an orthologue of B-sister genes GOA and ABS (Table 1). This


valuable starch and protein reserves. In the maturing caryopsis the fruit pericarp is reduced to just a few layers of largely collapsed cells and the cuticle-encased inner integument of the seed (Esau, 1960). It is the ‘shattering’, ‘threshing’, and ‘winnowing’ procedures that separate flower from fruit in cereal terminology i.e. separating the grain both from the pedicel at the base and from the adhering lemmae and palea (husk/ hull). Through milling, the bran (remaining pericarp, integuments, and aleurone) is further separated from the central endosperm. In considering fruit development and ripening in the context of the cereal grain, therefore, the surrounding floral tissues and seed contained within are of immense importance in describing the fruits journey. Genes controlling the encasing of ancestral maize (teosinte) kernels in a strong fruitcase (derived from the glume) and the adhering lemmae and palea tissues in hulled barley have been identified (Wang et al., 2005; Taketa et al., 2008). The hulled nature of the barley is quite unique in the cereals and so identifying the gene, a member of the AP2-EREBP gene family, controlling this feature will facilitate an understanding of why it has persisted (Taketa et al., 2008). There have been extensive advances in identifying the genetic basis of the shattering process since 2006, particularly in rice and more recently in sorghum. Furthermore, in some intriguing cases, the genes involved were related to those regulating fruit development and dehiscence in eudicots. Key shattering loci identified by QTL approaches in rice identified SH4 and qSH1, which encode trihelix and homeodomain transcription factors, respectively (Konishi et al., 2006; Li et al., 2006). qSH1 is the orthologue of RPL (REPLUMLESS) in Arabidopsis. Detailed analyses revealed that the genetic basis of qSH1 and RPL function in rice and Brassicaceae were analogous (Arnaud et al., 2011) where the cis element that modified qSH1 expression in rice was also affected in Brassica replums. Map-based cloning of an introgressed shattering locus identified SHAT1 as a third component in rice shattering, defining the abscission layer (AZ) in the rice rachilla (Zhou et al., 2012). SHAT1 is the orthologue of Arabidopsis AP2, a floral identity gene in the ABC model, but more recently also shown to have a role in negatively regulating RPL in replum development preventing overgrowth of the replum tissue in the carpel (Ripoll et al., 2011). The major domestication locus in wheat, Q, also encodes an AP2-like gene that contributes to various domestication traits including rachilla fragility and grain size (Simons et al., 2006). A YABBY family TF (YABBY2-like) was identified as an important determinant of shattering in sorghum, and also in rice and maize suggesting convergent evolution (Lin et al., 2012). This gene has not been assigned an explicit function in Arabidopsis, but its orthologue in tomato had been identified as the basis of the fas locus controlling locule number and fruit size difference between wild and cultivated tomato (Cong et al., 2008). In terms of the specification of the grass carpel itself, initial studies in rice found that, in contrast to eudicots, AG was not the master controller of carpel identity in cereals; although duplicated AG orthologues in grasses, and OsMADS3 and OsMADS58 in rice, affect carpel development, they did not specify the organ’s identity (Yamaguchi et al., 2006). This role


analysis illustrates the interplay of maternal and filial tissues in the grain’s development. In later stages of grain development forward genetics approaches (mutant screens) identified genes such as OPAQUE2 in maize as key regulators of storage protein gene expression (Hartings et al., 1989), and several TFs from the DOF, MYB, and BZIP gene families have been characterized in barley grains where they form complexes that regulate storage protein genes and genes responding to GA (gibberellic acid) and ABA (abscisic acid) hormones important in the maturation and germination process (Table 3). One of the few genes shown to specify particular tissues early in the grain development is ZmMRP1, a determinant of the endosperm transfer layer in maize (Gomez et al., 2009; Table 3). Even though different cereals have alternative transfer cells with functional homology, these may not be genetically homologous tissues in the evolutionary sense as they lack an identifiable orthologue of the MYB single repeat ZmMRP1 gene (Hands et al., 2012). Much of the molecular genetic research in cereals such as wheat has been hampered by the size and complexity of the genome (Shewry, 2009). Rice has formed the main monocotyledonous model system in terms of genomic resources, whereas maize provided an extensive resource and history of developmental genetics. However, rice as a semi-aquatic tropical species lacks many important temperate cereal traits. But with the recent and rapid advances in barley and wheat resources, including genome sequence (Brenchley et al., 2012; IBGSC, 2012), the stage is set for an exciting era in temperate cereal research generally, and grain research specifically. Before genome sequences became available for wheat and

barley, Brachypodium distachyon, had been developed as a new model system for the temperate cereals (IBI, 2010). Diploid B. distachyon offers one of the smallest genomes to be found in the entire grass family along with many other physical and phylogenetic attributes that make it suitable as a temperate grass model (Mur et al., 2011; Opanowicz et al., 2008). Logistically, the small stature, rapid life cycle, and simple growing conditions are particularly useful features for lab-based research where extensive or field-based growth facilities may not be available. Also, there is huge intrinsic value in having a wild uncultivated grass as a tractable model in comparative and ecological studies (Hands and Drea, 2012; Mur et al., 2011).

The cereal grain as a nutritional powerhouse Just as the BIA biosynthetic pathway of opium poppy has been teased apart, the starch, cell wall, and protein metabolic pathways in the grain that determine its uses/quality have also been characterized in depth. ‘Whole grains’ retain the remaining pericarp adhering to the seed (in the ‘bran’) and the associated nutritional value in terms of increased fibre that is usually removed in the ‘refined’ milling processes. Several different types of storage protein and the occurrence and relative amounts of these different proteins is an important and distinctive feature of different species and an important determinant of grain quality for bread-making etc. Amongst the Triticeae species and maize the prolamins form the dominant protein class and are highly significant to the processing and qualities of the grain, giving wheat flour is viscoelastic properties (Gibbon and Larkins, 2005; Shewry and Halford,


Fig. 4. Triticum aestivum (bread wheat). Left; photograph of a spike, spikelet, and grain (Hands et al., 2012). Right; caryopsis/grain in longitudinal profile and caryopsis/grain in transverse profile. A close-up of nucellar projection and adaxial endosperm is shown bottom right. Drawings based on Esau (1960), Drea et al., (2005), and Krishnan and Dayanandan (2003).

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Conclusions Cross-referencing Tables 1–3 and reviewing the research literature on comparative development shows that common genes are involved in similar but different aspects of fruit/seed development and maturation between eudicots and monocots, and that their roles are often modified in the context of the new morphology and evolutionary distance. OsMADS29/ ABS (B-sister) affect the development of the maternal tissues of the seed (nucellus and integuments) in rice and Arabidopsis (Nesi et al., 2002; Yin and Xue, 2012) while affecting starch accummulation in the grain. This link between central developmental regulators and commercial metabolic products is also evident in the case of AP2 and SHP orthologues in tomato, which affect the carotenoid accumulation in the ripening fruit (Chung et al., 2010; Vrebalov et al., 2009). Given that seed dispersal in a fleshy fruit like tomato involves the surrounding fruit tissues ripening and degrading, the role of these genes in affecting the rate of ripening could also be taken to constitute a role in fruit shattering/dehiscence and seed dispersal as it is in Arabidopsis and rice (in the case of the AP2 orthologue SHAT1; Zhou et al., 2012). AP2 is emerging as a ubiquitous determinant of various aspects of fruit development in eudicots and grasses (Ripoll et al., 2011, Ohto et al., 2009; Zhou et al., 2012, Simons et al., 2006; Houston et al., 2013). An intriguing case of conservation in the regulation of the RPL gene and its functional significance in seed dispersal has been demonstrated in Brassicaceae and rice (Arnaud et al., 2011). As for our MADS-box Cs and Ds, they will continue to be revisited many times and in many more species (Heijmans et al., 2012), whereas the FUL genes have already shown interesting diversification within Ranunculales (Pabon-Mora et al., 2012; 2013). Current and future work in cereals is already, and will continue to uncover patterns of conservation and diversification of regulatory gene function; in the basal eudicots there is an exciting programme of comparative fruit development in progress that promises to reveal much about development in the fruit and other tissues in this group of plants as well as extending what we know about the function of these important genes across the angiosperms. Eventually what we know about the genes regulating the fruits developmental programme from initiation though harvesting may be joined more seamlessly.

Acknowledgements The authors apologize for the huge volume of relevant research not cited in this review owing to limited space. Drawings in Figures 3 and 4 were done by Robert Harris based on cited work. Work on poppy and cereals in the lab is funded by The Leverhulme Trust and BBSRC, respectively.

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