Homeotic Genes and the ABCDE Model for Floral Organ Formation in Wheat

Abstract

Floral organ formation has been the subject of intensive study for over 20 years, particularly in the model dicot species Arabidopsis thaliana. These studies have led to the establishment of a general model for the development of floral organs in higher plants, the so-called ABCDE model, in which floral whorl-specific combinations of class A, B, C, D, or E genes specify floral organ identity. In Arabidopsis, class A, B, C, D, E genes encode MADS-box transcription factors except for the class A gene APETALA2. Mutation of these genes induces floral organ homeosis. In this review, I focus on the roles of these homeotic genes in bread wheat (Triticum aestivum), particularly with respect to the ABCDE model. Pistillody, the homeotic transformation of stamens into pistil-like structures, occurs in cytoplasmic substitution (alloplasmic) wheat lines that have the cytoplasm of the related wild species Aegilops crassa. This phenomenon is a valuable tool for analysis of the wheat ABCDE model. Using an alloplasmic line, the wheat ortholog of DROOPING LEAF (TaDL), a member of the YABBY gene family, has been shown to regulate pistil specification. Here, I describe the current understanding of the ABCDE model for floral organ formation in wheat.

1. Introduction

The ABCDE model for flower development proposes that floral organ identity is defined by five classes of homeotic genes, named A, B, C, D and E [1]. According to the floral quartet models of floral organ specification [2], the A- and E-class protein complex develop sepals as the ground-state floral organs in the first floral whorl, the A-, B- and E-class protein complex specify petals in the second whorl, the B-, C- and E-class protein complex specify stamens in the third whorl, and the C- and E-class protein complex specify carpels in the fourth whorl. Cloning of ABCDE homeotic genes in Arabidopsis showed that they encode MADS-box transcription factors except for the class A gene, APETALA2 (AP2) [3]. In Arabidopsis, the class A MADS-box gene is AP1 [4], the class B genes are AP3 and PISTILLATA (PI) [5,6], the class C gene is AGAMOUS (AG) [7], and the class D genes are SEEDSTICK (STK), SHATTERPROOF1 (SHP1) and SHP2 [8,9]. The D-class proteins interact in larger complex with the E-class proteins to specify ovule identity. In the Arabidopsis genome, four class E genes have been found, SEPALLATA1 (SEP1), SEP2, SEP3 and SEP4, which show partially redundant functions in identity determination of sepals, petals, stamens and carpels [10,11]. The diversification of MADS-box genes during evolution has contributed to the wide variation of flower forms in angiosperms [12]. Although they are not included in the conventional ABCDE model, the AGAMOUS LIKE 6 (AGL6)-clade genes AGL6 and AGL13, may play a role in floral organ formation, probably in ovule formation [13]. AGL6-clade genes comprise a sister clade of SEP genes and may share an E class function with SEP genes.

Grass species, such as rice (Oryza sativa), wheat (Triticum aestivum) and maize (Zea mays), form a unique reproductive inflorescence unit termed a spikelet [14,15]. The spikelet is comprised of florets and is encompassed by two small bract leaves (called glumes in wheat). Inflorescence development in wheat involves a series of stages: first, the inflorescence meristem produces a spikelet meristem as an axillary meristem; next, the spikelet meristem produces a floret meristem as an axillary meristem; finally, the floret meristem produces the floral organs (Figure 1, Figure 2) [16,17]. Development of the inflorescence in maize and rice is more complicated than in wheat because of the presence of additional axillary branch meristems: the tassel branch and spikelet pair meristem in maize, and the panicle branch meristem in rice [17,18]. In wheat, the spikelet is composed of florets that join the axis (rachilla) alternately on opposite sides, and is encompassed by two glumes (Figure 1, Figure 2). Each spikelet usually has six to eight florets, some of which, in apical positions, can be sterile due to hypoplasia. In each floret, the reproductive organs are enveloped by two leaf-like structures, a lemma and a palea. The lemma and palea are considered to have different origins. The lemma is a bract, which is a leaf subtending the axillary meristem of the spikelet axis; the palea is a prophyll, which is the first leaf formed by the axillary meristem [19]. An individual wheat flower contains one pistil, three stamens and two lodicules. The pistil, which is probably composed of three fused carpels, is the female part of the flower and consists of the ovary containing the ovule and two filamentous styles, each terminating with a feathery stigma. The stamen is composed of a filament and an anther containing pollen grains. Lodicules are attached to the ovary, and swell during anthesis forcing the lemma and palea apart to facilitate pollination of the stigma from the dehisced anther. There is evidence that the development of lodicules in rice and petals in Arabidopsis are regulated by a similar mechanism [20], suggesting that the lodicule was originally a modified petal. In summary, a palea, lodicules, stamens and a pistil are wheat floral organs developed in the whorl 1, 2, 3, and 4, respectively. Analysis of ABCDE genes in monocot species such as rice suggests that the ABCDE model might equally apply to monocots [18,21]. Here, we focus on application of the ABCDE model to flower development in wheat.

Figure 1. Wheat inflorescences and floral organs. (a) Developing young spike at the floret differentiation stage. The spikelet primordium (Spp) is indicated. Scale bar = 1 mm; (b) The wheat inflorescence (spike, ear, or head) is composed of spikelets (Sp) attached at the nodes of a zigzag rachis (Rs). Scale bar = 2 cm; (c) A spikelet that has been removed from the rachis. The spikelet consists of multiple (usually six to eight) florets attached at the rachilla (Ra). Two small bract leaves called glumes (Gl) enclose the spikelet. Scale bar = 1 cm; (d) A magnified image of an opened floret. In the floret, the reproductive organs, pistil (Pi) and stamens (St) are enveloped by two leaf-like structures, the lemma (Le) and the palea (Pa). The lemma and palea have been separated to make the reproductive organs visible in the figure. Scale bar = 2 mm; (e) An individual flower containing one pistil (Pi), three stamens (St) and two lodicules (Lo). The palea (Pa) is also indicated. In this figure, the pistil, stamens, lodicules and palea have been removed from the rachilla. Scale bar = 2 mm; (f) A flower from a plant of the pistillody line. The stamens are transformed into pistil-like structure (Pst) with stigmas. Scale bar = 2 mm.

Figure 1. Wheat inflorescences and floral organs. (a) Developing young spike at the floret differentiation stage. The spikelet primordium (Spp) is indicated. Scale bar = 1 mm; (b) The wheat inflorescence (spike, ear, or head) is composed of spikelets (Sp) attached at the nodes of a zigzag rachis (Rs). Scale bar = 2 cm; (c) A spikelet that has been removed from the rachis. The spikelet consists of multiple (usually six to eight) florets attached at the rachilla (Ra). Two small bract leaves called glumes (Gl) enclose the spikelet. Scale bar = 1 cm; (d) A magnified image of an opened floret. In the floret, the reproductive organs, pistil (Pi) and stamens (St) are enveloped by two leaf-like structures, the lemma (Le) and the palea (Pa). The lemma and palea have been separated to make the reproductive organs visible in the figure. Scale bar = 2 mm; (e) An individual flower containing one pistil (Pi), three stamens (St) and two lodicules (Lo). The palea (Pa) is also indicated. In this figure, the pistil, stamens, lodicules and palea have been removed from the rachilla. Scale bar = 2 mm; (f) A flower from a plant of the pistillody line. The stamens are transformed into pistil-like structure (Pst) with stigmas. Scale bar = 2 mm.

Figure 2. Schematic illustrations of the phytomeric structures of the wheat inflorescence. The spikelets are arranged as two opposite rows of lateral branches from the main axis (rachis). Each spikelet is composed of florets joined at the axis (rachilla) alternately on opposite sides, and enclosed by two glumes. Each floret is composed of a lemma, a palea, two lodicules, three stamens and a pistil. gl, glume; le, lemma; pa, palea; lo, lodicule; st, stamen; pi, pistil.

Figure 2. Schematic illustrations of the phytomeric structures of the wheat inflorescence. The spikelets are arranged as two opposite rows of lateral branches from the main axis (rachis). Each spikelet is composed of florets joined at the axis (rachilla) alternately on opposite sides, and enclosed by two glumes. Each floret is composed of a lemma, a palea, two lodicules, three stamens and a pistil. gl, glume; le, lemma; pa, palea; lo, lodicule; st, stamen; pi, pistil.

2. Summary of ABCDE Model in Rice

2.1. Rice Class A Genes

Three AP1-like MADS-box genes have been identified in the rice genome, namely, OsMADS14/RAP1B, OsMADS15/RAP1A and OsMADS18, which are all derived from the FRUITFULL (FUL) lineage rather than AP1 as in Arabidopsis [22]. Studies on transgenic plants suggested that OsMADS14 is involved in promoting flowering and in determining the identity of the floral meristem [23]. Interestingly, analysis of the OsMADS15 mutant degenerative palea (dep) indicated that OsMADS15 plays a role in palea formation [24]. On the basis that the palea of rice, rather than the lemma, is evolutionarily identical with the sepal of Arabidopsis, then OsMADS15 is likely to be a rice class A gene. However, the dep mutation does not cause defects in the lodicules [24], suggesting that lodicule specification is controlled by another class A gene, an AP2-like gene. It has been reported that overexpression of micro RNA miR172, a negative regulator of AP2, results in the conversion of lodicules into the palea marginal region in transgenic rice plants [25]. Recently, two AP2-like genes, SUPERNUMERARY BRACT (SNB) and Os INDETERMINATE SPIKELET1 (OsIDS1), were identified in rice to be required for lodicule development [26]. SNB and OsIDS1 are positively regulated by another AP2-like gene, MULTI-FLORET SPIKELT1 (MFS1) [27]. Furthermore, these rice AP2-like genes determine inflorescence architecture by regulating changes in spikelet meristem fate.

2.2. Rice Class B Genes

OsMADS2 and OsMADS4 have been reported to be the rice orthologs of PI and to have been generated by an ancient gene duplication event [28]. RNAi suppression of OsMADS2 results in homeotic change to lodicules but stamens still develop normally [29]. By contrast, RNAi suppression of OsMADS4 does not induce any alterations to either lodicules or stamens [30], although simultaneous loss-of-function in both OsMADS2 and OsMADS4 results in conversion of lodicules to palea-like organs and stamens to carpel-like organs [30]. These observations indicate that OsMADS2 plays a more important role than OsMADS4 in lodicule specification, and that OsMADS2 and OsMADS4 have an equal function in stamen formation.

OsMADS16 /SUPERWOMAN1 (SPW1) is the sole AP3 ortholog in the rice genome [31]. A yeast two-hybrid assay indicated that OsMADS16 interacts with both OsMADS2 and OsMADS4 [32]. Loss-of-function of OsMADS16 causes the same phenotype as RNAi-mediated simultaneous suppression of OsMADS2 and OsMADS4, i.e., the conversion of lodicules and stamens into palea-like and carpel-like organs, respectively [31]. Overall, these findings indicate that the two PI-like genes, OsMADS2 and OsMADS4, and the AP3-like gene, OsMADS16, are class B genes in rice.

2.3. Rice Class C Genes

The duplicated class C genes in rice, OsMADS3 and OsMADS58, have been reported to show partial conservation of function with the Arabidopsis class C gene, AG. Mutant and transgenic analyses indicated that OsMADS3 predominantly regulates stamen identity and prevents lodicule development and that OsMADS58 regulates floral meristem determinacy and normal carpel morphogenesis [33]. However, a recent study on OsMADS3 and OsMADS58 mutants suggested that the two genes redundantly mediate the C-function and, together with OsMADS13 (a class D gene), are important for floral meristem determinacy [34]. Furthermore, it is recently reported that the two class C genes interacts with OsMADS16 (a class B gene) in suppressing indeterminate growth within the floral meristem [35]. Interestingly, carpel identity in rice is determined by a YABBY gene named DROOPING LEAF (DL) [31,36].

2.4. Rice Class D Genes

Analyses of expression and of protein-protein interactions suggested that the rice class D gene OsMADS13 is involved in specifying ovule identity [37,38]. Recent mutation and los-of-function studies of OsMADS13 revealed that it controls ovule specification [39,40]. Mutation of OsMADS21, a paralog of OsMADS13, does not result in any additive ovule defect, indicating that OsMADS21 has lost its ability to determine ovule identity [34,39].

2.5. Rice Class E Genes

The class E genes of rice belong to two clades, the SEP-clade and the LOFSEP-clade [41]. In the SEP-clade, OsMADS7/OsMADS45 and OsMADS8/OsMADS24 show high sequence similarity to Arabidopsis SEP genes [41]. Simultaneous suppression of OsMADS7 and OsMADS8 causes severe meristic and homeotic effects in the inner three floral whorls; in particular, lodicules are transformed into lemma/palea-like structures [42].

The LOFSEP-clade contains OsMADS1/LEAFY HULL STERILE 1 (LHS1), OsMADS5/OSM5, and OsMADS34/PANICLE PHYTOMER 2 (PAP2) [43]. Mutation of OsMADS1 in rice produces the leafy hull sterile 1 (lhs1) phenotype that has a leaf-like lemma and palea, and lemma/palea-like lodicules [44]. Furthermore, knockdown of OsMADS1 induces the transformation of the lemma into a glume-like structure [45]. These results indicate that OsMADS1 functions in lemma and palea differentiation. In contrast with OsMADS1, mutation of OsMADS34 developed altered inflorescence morphology with altered numbers of primary and secondary branches [46]. These indicate that OsMADS34 and OsMADS1 play important functions in specifying the inflorescence and spikelet. Recently, it was reported that OsMADS34 acts in the shoot apical meristem together with the three AP1/FUL-like genes, OsMADS14, OsMADS15 and OsMADS18, to specify the identity of the inflorescence meristem [47]. Simultaneous silencing of LHS1, OsMADS5, OsMADS7, and OsMADS8 is sufficient to transform all floral organs, except the lemma, into leaf-like structures indicating that the four genes act in concert to provide a class E function in rice [42].

The rice AGL6-clade gene, OsMADS6/MOSAIC FLORAL ORGANS 1 (MFO1), regulates floral organ identity, suggesting that it also has an E class function [48,49]. Another AGL6-clade gene, OsMADS17, has a minor but redundant function with that of MFO1. Recently, mutant analyses indicated that OsMADS6 plays synergistic roles in floral organ specification with class B, C, D genes and with DL [50]. Furthermore, a null allele of OsMADS6 exhibited transformation of floral organs except for lemma into lemma-like organs [51], indicating that OsMADS6 acts as a critical regulator for floral organ formation.

3. Pistillody, Homeotic Transformation of Stamens into Pistil-like Structures, in the Alloplasmic Wheat Line

To investigate the effects of cytoplasm from wild relatives of common wheat (Triticum aestivum) on floral development, cytoplasmic substitution (alloplasmic) lines have been produced by recurrent backcrossing [52,53]. In an alloplasmic line in which Aegilops crassa cytoplasm has been introduced into the wheat cultivar (cv.) Norin 26 (N26), male sterility occurs under long-day conditions (>15 h light period) due to pistillody, the homeotic transformation of stamens into pistil-like structures (Figure 1) [54]. This phenomenon was named photoperiod-sensitive cytoplasmic male sterility (PCMS) and has been extensively investigated to assess its value to hybrid wheat breeding [55]. In contrast to N26, the wheat cv. Chinese Spring (CS) does not show pistillody when Ae. crassa cytoplasm is introduced; the absence of an effect is due to a single dominant gene (designated Rfd1) located on the long arm of chromosome 7B [56]. The role of Rfd1 has been investigated by a loss-of-function analysis in an alloplasmic line of CS with ditelosomy of chromosome 7BS, i.e., lacking the long arm of chromosome 7B, and with Ae. crassa cytoplasm {(cr)-CSdt7BS}. These plants showed pistillody indicating that the absence of Rfd1 induces the phenotype irrespective of photoperiod. By contrast, CS plants with ditelosomy of 7BS but with a normal cytoplasm (CSdt7BS) form normal stamens [57]. These results indicate that pistillody is induced by factor(s) in the Ae. crassa cytoplasm, presumably from mitochondrial gene(s), and that the nuclear Rfd1 gene prevents the deleterious effects of the cytoplasm. PCMS in the alloplasmic lines of N26 suggests the presence of an Rf gene that functions under short-day conditions. One candidate for the Ae. crassa cytoplasmic factor causing pistillody in alloplasmic wheat is the mitochondrial gene orf260 [58]. It is also possible that retrograde (mitochondrion to nucleus) signaling via a protein kinase and calmodulin-binding protein may be involved in pistillody induction [59,60]. In the alloplasmic line, an ectopic ovule differentiates in the pistil-like stamens [54,57]. The pistillody line (cr)-CSdt7BS and the corresponding normal line CSdt7BS are useful for investigating the molecular mechanism of the homeotic change of stamens into pistil-like structures with an ectopic ovule induced by a cytoplasmic factor, and for identification of class BCD MADS-box genes. The functions of wheat class BCD MADS-box genes in detail would be examined by the transgenic studies.

4. Pistillody Reveals the Function of Class BCD MADS-Box Genes in Wheat

4.1. Wheat Class B Genes

In the ABCDE model, the loss-of-function in class B MADS-box genes (AP3 and PI in Arabidopsis) results in pistillody, the homeotic transformation of stamens into carpel/pistil-like structures. The highly homologous wheat AP3-type genes, TaMADS#51 and TaMADS#82, were the first Class B MADS-box genes to be identified [61]. Bread wheat is a hexaploid with the genomic constitution AABBDD in which each genome originated from a different ancestral species. The A genome is believed to derive from T. urartu, the B genome from Aegilops speltoides or another species in the Sitopsis section, and the D genome from Ae. tauschii [62]. Allopolyploidization leads to the generation of duplicated homoeologous genes (homoeologs) and, consequently, the hexaploid wheat genome contains triplicated homoeologs derived from the three ancestral diploid species. TaMADS#51 and TaMADS#82 are wheat homoeologs of the AP3 ortholog (wheat APETALA3: WAP3) and are located on chromosomes 7B and 7D, respectively. A northern blot analysis showed that expression of WAP3 is restricted to young spikes at the floral organ developing stage, suggesting that WAP3 functions in floral organ formation [61]. The level of expression of WAP3 is reduced in the pistillody line compared to the normal line [57]. WAP3 has also been called TaAP3 [63].

Two PI-type genes have been identified in wheat, namely WPI1 (wheat PISTILLATA1) and WPI2 [64]. A phylogenetic analysis using the deduced amino acid sequences indicated that WPI1 and WPI2 are orthologs of the rice PI-type genes OsMADS4 and OsMADS2, respectively. WPI1 and WPI2 have also been called TaPI-1 and TaPI-2/TaAGL26, respectively [63,65].

An in situ expression analysis showed that WPI and WAP3 are expressed in the primordia of the stamen and lodicule in the normal wheat line; however, no transcripts were detectable in the pistil-like stamens of the pistillody line [64]. This finding indicates that pistillody results from a deficit of WPI and WAP3 expression in whorl 3, suggesting that these genes have a class B function.

4.2. Wheat Class C Genes

The AG orthologs of wheat, WAG1 (wheat AGAMOUS1) and WAG2, were identified as class C genes [66,67]. The level of transcription of WAG genes is low at the early stages of initiation of floral organ primordia and at its highest at the booting to heading stages. An in situ expression analysis indicated that WAG genes are associated with pistil and pistilloid stamen formation in the alloplasmic line [68]. A phylogenetic analysis using the deduced amino acid sequences showed that WAG1 and WAG2 are orthologs of the rice AG-type genes, OsMADS58 and OsMADS3, respectively [67,69]. WAG1 and WAG2 are also called TaAG-1 and TaAG-2/TaAGL39, respectively [63,65].

4.3. Wheat Class D Genes

Two studies in wheat have identified five genes, TaAGL2, TaAGL9, TaAGL31, TaAG-3A and TaAG-3B as candidate orthologs of the rice class D gene, OsMADS13 [63,65]. Subsequent sequence analyses showed that TaAG-3A is identical with TaAGL9, and TaAG-3B is identical with TaAGL2. Furthermore, TaAGL2, TaAGL9 and TaAGL31 show very high sequence similarity suggesting that may be homoeologous. These wheat orthologs of Arabidopsis STK have been renamed as WSTK (wheat SEEDSTICK) [68].

In alloplasmic wheat, ectopic expression of the class D gene WSTK occurs in the adaxial region of pistil-like stamens and ectopic ovule primordia are initiated in these regions [68]; this suggests that WSTK expression is involved in ectopic ovule formation in pistil-like stamens. In Arabidopsis, STK functions in ovule development by an interaction with the class C-lineage MADS-box genes, AG, SHP1 and SHP2, which is mediated by the class E gene SEP3 [8]. In the pistil-like stamens of alloplasmic wheat, ectopic expression of the class C MADS-box genes, WAG1 and WAG2, and the class D gene WSTK is induced [68]. Furthermore, WSTK protein forms a complex with the class E protein, WSEP, but not with the class C proteins WAG1 and WAG2 [68]. These facts suggest that WSTK has a class D function in wheat, similar to STK in Arabidopsis.

4.4. Wheat DLOOPING LEAF Gene, TaDL

In rice, carpel (pistil) specification is regulated by the DROOPING LEAF (DL) gene that encodes a YABBY transcription factor [36]. TaDL, a DL ortholog in wheat, was identified by homology screening [70]. In situ expression analysis in the pistillody line showed that TaDL is expressed in the primordia of pistil-like stamens as well as in the pistil. This suggests that TaDL functions in specification of the pistil. Together with the observation that class B genes are not detected in the primordia of pistil-like stamens [64], these facts suggest mutual repression between TaDL and class B genes.

5. Other Homeotic Genes in Wheat

5.1. Wheat Class E Genes

With regard to SEP-like genes, two MADS-box genes, WSEP (wheat SEPALLATA) and WLHS1 (wheat LEAFY HULL STERILE 1) have been identified in wheat [71]. Phylogenetic analysis showed that WSEP clusters in the same group as OsMADS24 and OsMADS45. In situ hybridization experiments showed that WSEP is expressed in the inner three whorls (lodicules, stamens and pistils) at the floral organ differentiation stage. Interestingly, after floral organ identities have been determined, strong expression of WSEP is observed in the palea, suggesting that WSEP genes are not only involved in floral organ differentiation but also in their subsequent development. The palea-specific expression was also observed in rice OsMADS6 (an AGL6-like gene), suggesting the unique role of class E gene in grasses [72]. Yeast two- and three-hybrid analyses indicated that WSEP forms a complex with wheat class B and C genes [71], in a similar fashion to Arabidopsis SEP3 [73].

In addition to WSEP, TaMADS1 has been identified and characterized as a wheat class E gene [74]. A phylogenetic study indicated that WSEP is an ortholog of rice OsMADS45 and that TaMADS1 corresponds to OsMADS24; this suggests that SEP orthologs have diverged into two groups in monocot species [71]. Transgenic Arabidopsis plants over-expressing TaMADS1 show early flowering and terminal flower formation [74]. Although protein-protein interactions involving TaMADS1 and wheat class B or C genes have not yet been examined, TaMADS1 may have a similarity as WSEP, because over-expression of WSEP in Arabidopsis causes early flowering and terminal flower formation [71].

Based on phylogenetic studies, WLHS1 is a wheat ortholog of OsMADS1 [71], a member of LOFSEP-clade. Transcripts of WLHS1 accumulate at high levels in the glume, lemma and palea, and at a low level in the pistil and stamen. It has been reported that OsMADS1-like gene expression in inflorescences varies among grasses such as Sorghum bicolor, Chasmanthium latifolium, Avena sativa, and Pennisetum glaucum [75]. The differences in the expression patterns of OsMADS1-like genes in wheat and other grass species may be associated with differences in the structures of their respective inflorescences.

In wheat, five genes, TaMADS#12, TaAGL37, TaAGL6-1A, TaAGL6-1B and TaAGL-1C, have been identified as candidate orthologs of AGL6-like genes [61,63,65]. TaMADS#12 and TaAGL6-1B are identical, as are TaAGL37 and TaAGL6-1A; this suggests that these genes are homoeologs. The function of wheat AGL6-like genes has yet to be ascertained.

5.2. Wheat Class A Genes

Arabidopsis has two class A genes, AP1 and AP2. The AP1 MADS-box gene functions in the specification of floral meristem identity and in the determination of sepal development. There are two other AP1-like genes, FRUITFULL (FUL) and CAULIFLOWER (CAL), which have redundancy of function in specification of floral meristem identity with AP1 [76]. Sequence analysis of AP1-like genes in monocots suggests that they only have FUL-like proteins, in contrast to dicot species, which have AP1, FUL and CAL proteins [22].

The grass family genome has three paralogs of AP1/FUL-like genes, namely, FUL1 (corresponding to VERNALIZATION1 (VRN1) in wheat), FUL2 and FUL3, which are all derived from the FUL lineage [22]. Wheat FUL1, WFUL1/VRN1, has no class A function but acts in phase transition from vegetative to reproductive growth [77,78,79,80]. A phylogenetic analysis using the deduced amino acid sequences showed that WFUL1, WFUL2 and WFUL3 are orthologs of the rice AP1-type genes, OsMADS14, OsMADS15 and OsMADS18, respectively [81].

In young spikes, expression of WFUL2 is greatly reduced in stamens and cannot be detected in pistils, whereas WFUL1/VRN1 and WFUL3 are expressed in all floral organs [81], suggesting that WFUL2 has a different function in the outer floral organs (lemma and palea) compared to the inner floral organs (stamen and pistil). Yeast two- and three-hybrid analyses showed that WFUL2 interacts with class B and class E proteins [81]. In combination with the expression analyses, these observations suggest that WFUL2 specifies the identity of the outer floral organs in the wheat floret. In rice, both FUL1 and FUL2 proteins (OsMADS14 and OsMADS15, respectively) interact with a class E protein (OsMADS1/LHS1) [82], suggesting that the diversification of function between FUL1 and FUL2 detected in wheat has not occurred in rice. Especially, it is notable that wheat FUL1 (WFUL1/VRN1) has important role at leaves as well as at shoot apex in flowering [81]. Expression and protein-protein interaction studies suggested that WFUL2 in wheat has a class A function in development of the outer floral organs (lemma and palea) in combination with class B and class E MADS-box genes [81].

The wheat Q gene has been identified as an AP2-like gene [83]. The q allele confers a ‘speltoid’ spike phenotype that is characterized by a loosely formed head structure with elongated rachis and non-free-threshing seed. A phylogenetic analysis found that Q is not orthologous to Arabidopsis AP2; rather, another AP2-like gene, TaAP2, is the AP2 ortholog [84]. The barley AP2 ortholog, HvAP2/Cly1 is associated with lodicule development [85], suggesting that TaAP2 in wheat functions in floral organ formation, especially in lodicule development. Together with the observations in rice AP2-like genes [26,27], these findings may imply that the AP2-like gens in grasses have common function in floral organ formation.

6. Wheat ABCDE Model, Complicated Homoeologous Gene Interaction

The wheat ABCDE model for floral organ formation is illustrated in Figure 3. The relationships of homeotic genes among Arabidopsis, rice and wheat are shown in Table 1. As mentioned earlier, wheat is an allohexaploid species with the genome constitution AABBDD. Consequently, the hexaploid wheat genome contains triplicated homoeologs derived from the three ancestral A, B and D genomes. There are three possible evolutionary fates for homoeologs in polyploids: functional diversification, gene silencing, and retention of original or similar function [86]. Functional diversification of homoeologs is one of the important factors in the evolutionary success of polyploid species [87].

With regard to class E genes, analyses of gene structure, expression patterns and protein functions showed no evolutionary changes to the WSEP homoeologs. In contrast, the three WLHS1 homoeologs show genetic and epigenetic alterations [71]. The A genome WLHS1 homoeolog (WLHS1-A) has a large deletion in the region of the K domain sequence. Data from a yeast two-hybrid analysis and a transgenic experiment indicated that the WLHS1-A protein does not have a function in floral development. WLHS1-B and WLHS1-D, located in the B and D genomes, respectively, have the complete MADS-box gene structure; however, WLHS1-B is effectively silenced by epigenetic regulation. Consequently, of the three homoeologs, only WLHS1-D functions in hexaploid wheat.

The example of the WLHS1 genes indicates the possibility that homoeologs of each homeotic gene may be differentially regulated in wheat spike formation. Floral homeotic MADS domain proteins interact in floral tissue as proposed in the “floral quartet” model, in which a tetramer of MADS domain proteins functions in specification of floral organ identity [2,73]. The complex homoeologous gene interactions are probably associated with morphological, physiological and ecological diversification among different ploidy levels. Polyploid wheat must be a good model for investigating this point [88].

Figure 3. The ABCDE model of floral organ formation in wheat. In contrast to the Arabidopsis ABCDE model, the wheat ABCDE model involves duplicated genes for class B (PI-like) and class C (AG-like) functions. Furthermore, class E genes are divided into two groups, WSEP and WLHS1, with sub-functionalization. A YABBY gene TaDL specifies the pistil (carpel) identity. The pistillody line has been valuable for constructing the ABCDE model in wheat. The wheat ABCDE model is similar to that of rice except for the class A genes. The current wheat ABCDE model indicates that class B and TaDL proteins show mutual suppression, which was suggested from analysis of a pistillody line. The mutual suppression between class A and C genes is also postulated here. The wheat ABCDE model probably functions through complex homoeologous gene interactions.

Figure 3. The ABCDE model of floral organ formation in wheat. In contrast to the Arabidopsis ABCDE model, the wheat ABCDE model involves duplicated genes for class B (PI-like) and class C (AG-like) functions. Furthermore, class E genes are divided into two groups, WSEP and WLHS1, with sub-functionalization. A YABBY gene TaDL specifies the pistil (carpel) identity. The pistillody line has been valuable for constructing the ABCDE model in wheat. The wheat ABCDE model is similar to that of rice except for the class A genes. The current wheat ABCDE model indicates that class B and TaDL proteins show mutual suppression, which was suggested from analysis of a pistillody line. The mutual suppression between class A and C genes is also postulated here. The wheat ABCDE model probably functions through complex homoeologous gene interactions.

Table 1. The relationships of homeotic genes among Arabidopsis, rice and wheat.

Class	Clade	Arabidopsis	Rice	Wheat
class A		AP1	OsMADS14/RAP1B	WFUL1/VRN1
			OsMADS15/RAP1A	WFUL2
			OsMADS18	WFUL3
		AP2	SNB	TaAP2
			OsIDS1	Q
			MFS1
class B		AP3	OsMADS16/SPW1	WAP3/TaAP3 *
		PI	OsMADS2	WPI2/TaPI-2/TaAGL26
			OsMADS4	WPI1/TaPI-1
class C		AG	OsMADS3	WAG2/TaAG-2/TaAGL39
			OsMADS58	WAG1/TaAG-1
class D		STK	OsMADS13	WSTK **
		SHP1, 2
class E	SEP	SEP1, 2, 3, 4	OsMADS7/OsMADS45	WSEP
			OsMADS8/OsMADS24	TaMADS1
	LOFSEP		OsMADS1/LHS1	WLHS1
			OsMADS5/OsM5
			OsMADS34/PAP2
	AGL6	(AGL6)	OsMADS6/MFO1	TaAGL6 ***
			OsMADS17
other		(CRC)	DL	TaDL

* TaMADS#51 and TaMADS#82 are two of three homoeologs of WAP3; ** TaAGL2/TaAG-3B, TaAGL9/TaAG-3A and TaAGL31 are homoeologs of WSTK; *** TaAGL6-1A/TaAGL37, TaAGL6-1B/TaMADS#12 and TaAGL6-1C are homoeologs of TaAGL6.

Table 1. The relationships of homeotic genes among Arabidopsis, rice and wheat.

Class	Clade	Arabidopsis	Rice	Wheat
class A		AP1	OsMADS14/RAP1B	WFUL1/VRN1
			OsMADS15/RAP1A	WFUL2
			OsMADS18	WFUL3
		AP2	SNB	TaAP2
			OsIDS1	Q
			MFS1
class B		AP3	OsMADS16/SPW1	WAP3/TaAP3 *
		PI	OsMADS2	WPI2/TaPI-2/TaAGL26
			OsMADS4	WPI1/TaPI-1
class C		AG	OsMADS3	WAG2/TaAG-2/TaAGL39
			OsMADS58	WAG1/TaAG-1
class D		STK	OsMADS13	WSTK **
		SHP1, 2
class E	SEP	SEP1, 2, 3, 4	OsMADS7/OsMADS45	WSEP
			OsMADS8/OsMADS24	TaMADS1
	LOFSEP		OsMADS1/LHS1	WLHS1
			OsMADS5/OsM5
			OsMADS34/PAP2
	AGL6	(AGL6)	OsMADS6/MFO1	TaAGL6 ***
			OsMADS17
other		(CRC)	DL	TaDL

* TaMADS#51 and TaMADS#82 are two of three homoeologs of WAP3; ** TaAGL2/TaAG-3B, TaAGL9/TaAG-3A and TaAGL31 are homoeologs of WSTK; *** TaAGL6-1A/TaAGL37, TaAGL6-1B/TaMADS#12 and TaAGL6-1C are homoeologs of TaAGL6.