**5. Functional Conservation and Diversification between Distinct Floral Specification Systems**

If it is assumed that all angiosperms have homologous reproductive organs, then divergent angiosperm groups may have a single ancestral flower specifying genetic mechanism. The modified floral organ identity model (ABCDE) in Arabidopsis suggests occurrence of genetic interactions among floral homeotic genes. The same model can be used to interpret molecular control of inflorescence identity in other crop plants including cereals [37]. High-throughput forward and reverse genetic approaches have led to the identification, cloning, and functional characterization of several genes involved in the regulation of floral development especially in grasses. Interestingly, most of these genes exhibit highest sequence similarites and share expression patterns and functional properties with those of eudicot A, B, C, D, and E floral homeotic genes. However, some grass-specific floral regulators have also been identified that do not have eudicot homologs and perform distinct functions in grass floral development. This review integrates current knowledge of floral organ identity genes in an attempt to adopt the eudicot floral organ identity model to other crop species. Considering grass organ-identity models illustrated in Figure 2, it is apparent that further research is needed to functionally characterize maize, Brachypodium, and wheat MADS-box genes to manipulate for crop grain yield improvement.

Prior to the discovery of loss-of-function mutants, gene function was usually examined through sequence conservation and expression pattern comparisons with already characterised genes. Due to genome complexities and difficulties in the use of modern genetic approaches in grasses, very little was known about the role of MADS-box genes in controlling spikelet and floret development. However, recent studies have provided new insights into the conservation of class (A)-gene function among eudicots and cereals [65]. The mutational analyses of *AP1*/*FUL* like genes in rice and demonstrated that in addition to their role in floral meristem identity, they also influenced the specification of palea and lodicule identities. As these grass-specific organs are thought to be homologous to eudicot's sepals and petals, these divergent groups may share a conserved nonreproductive floral organ specification system.

Comparison of all the proposed models indicates a partially conservative, partially-diverse floral regulation among grasses and higher eudicots. Based on studies in model eudicots, gene expression patterns and mutant phenotypes appear to be consistent with functional predictions. This is essentially true for class B, C, and D MADS-box genes. Like eudicots, the functions of class B and C genes have diverged in grasses due to duplication and subfunctionalization of separate genes. For example, rice *PI*-like class B genes show unequal redundancy in their function. Individual mutant analysis of *OsMADS2* indicates the homeotic conversion of lodicules without affecting stamens, whereas *OsMADS4* shows no alteration in lodicules and stamens. Additionally, double mutants of both genes show the conversion of lodicule and stamen into palea and carpel-like structures, suggesting an equal role of both genes in stamen identity. However, *OsMADS2* is more important than *OsMADS4* in lodicule identity. Mutant analysis of maize *PI* orthologs also indicated that *ZMM16*/*STS1* can compensate *zmm18*/*29* reduction while specifying class B gene functions. However, *ZMM18*/*29* cannot compensate for *zmm16*/*sts1* reduction. Similar roles may be speculated for Brachypodium and wheat *PI* orthologs as *BdMADS16* and *WPI1* show sequence similarity with *OsMADS4* and *ZMM18*, respectively, while *BdMADS20* and *WPI2* have greater similarity with respect to *OsMADS2* and *ZMM16* (Figure 3).

Subfunctionalization of duplicated genes was also observed among grass related class C genes. According to Yamaguchi et al. [98], *OsMADS58* plays a major role in floral meristem determinacy with minor effects on floral organ identity; whereas *OsMADS3* has a dominant role in stamen identity with a minor role in meristem determinacy. Similar findings were reported by Dreni et al. [92], in which *OsMADS3* showed to regulate stamen identity compared with *OsMADS58*. Severe defects were obsereved in *osmads3* mutant flowers, whereas most of the *osmads58* mutant flowers were indistinguishable from the wild type flowers. Likewise, the maize orthologs *ZAG1* and *ZMM2* exhibit functional diversification and are homologs to *OsMADS58* and *OsMADS3*, respectively. *ZAG1* mutant showed loss of floral meristem determinacy and have a minor role in stamen and carpel identity. By contrast, *ZMM2* has yet to be characterized for floral organ identity. In Brachypodium, *BdMADS14* and *BdMADS18* also indicated overlapping but diverse expression patterns as *BdMADS14* highly expressed in stamens only compared to *BdMADS18* that strongly expressed both in stamens and carpels. Based on orthologous relationships, Brachypodium and maize class C genes have overlapping but nonidentical functions, similar to rice genes. In contrast, the wheat class C genes *WAG-1* and *WAG-2* are involved in ectopic ovule formation and homeotic conversion of stamens into pistil- like structures and are orthologous to *OsMADS58* and *OsMADS3,* respectively. Knockdown mutants of *WAG* genes will help elucidate their function in floral organ identity. Mutations in rice and maize class C genes result in homeotic conversion of stamens and carpels into lodicules and paleae-like organs, respectively. Similarly, class C gene mutations in Arabidopsis caused homeotic conversion of both reproductive organs, with few exceptions. Mutations of these genes in wheat result in ectopic ovule formation and homeotic conversion of stamens into pistil-like structures. Thus, diverse carpel specification systems operate in these two divergent groups.

Genetic interactions between rice class C and D MADS-box and non-MADS-box (*DL*) genes provided new insights into the partially conservative and partially diversified mechanisms regulating floral development in eudicots and grasses. Li et al. [159] studied double mutants of *OsMADS3*, *OsMADS13*, and *DROOPING LEAF* (*DL*) to investigate their role in floral development. Termination

of floral meristem determinacy and several carpelloid structures were observed in *osmads3*/*osmads13* double mutants, while noteably their single mutants lacked these alterations at all. Furthermore, gene expression and protein–protein interaction analyses revealed that both C and D class genes neither regulate nor interact at the transcription or protein levels, suggesting that class E genes would mediate their interaction to synergistically control the termination of floral meristem and ovule identity. These obervations support the notion that grasses have retained their class C and D gene functions, despite undergone duplication and subfunctionalization. Dramatically, double mutants of *osmads3*/*dl* showed no *AG* activity, with production of lodicule-like structures within the fourth whorl and the termination of floral meristem determinacy. Mutual suppression was also absent and normal expression patterns were observed for *OsMADS3* and *DL* genes in ectopic stamens of *dl* flowers and *osmads3* mutants, respectively, suggesting a redundant role for both genes in floral meristem termination. In contrast, single and double mutants of *OsMADS13* and *DL* suggest that *DL* is epistatic to *OsMADS3*, and both have identical roles in ovule identity. More recently, the role of ovule in cereal grain development has been briefly reviewd [172]. The phenotype of single *dl* mutants was identical to that of double *osmads13*/*dl* mutants. The *dl* mutants lacked *OsMADS13* expression, whereas single mutant of *osmads13* showed abundance of *DL* transcripts, indicating direct or indirect *OsMADS13* regulation by *DL*. Dreni et al. [92] investigated genetic interactions between rice class C and D genes (*OsMADS13* and *OsMADS58*) through single- and multiple-gene mutants. As expected, *osmads13*/*58* double mutants showed accumulation of lodicule and palea-like organs in the third and fourth whorls accompanied by loss of floral organ identity and the triggering of floral meristem indeterminacy due to reduced *AG* activity. These observations suggest that a highly conserved class C gene functional mechanism exists in grasses despite partial subfunctionalization among duplicated genes. These results led to the proposition that *DL* lacks class C gene functional activity and cannot specify carpel identity alone, requiring both *OsMADS3* and *OsMADS58*. Although these findings provide new insights into the floral development process, further examination of single and multiple mutants of class B, C, D, and *DL* genes will be required to elucidate the roles of MADS-box and *DL* genes in floral organ specification.

The function of class E genes, particularly the *SEP*-like, are somewhat conserved among eudicots and grasses. In rice, detailed spatial and temporal mRNA expression studies, protein interaction patterns, and mutant analysis indicated a consistent role for *SEP*-like genes in floral meristem and organ identity specification [104], since divergence from eudicots. Mutant phenotypes of Arabidopsis *SEP* redundant genes (*SEP1*/*2*/*3*/*4*) and rice *OsMADS1*, *OsMADS7*, and *OsMADS8* genes indicated a redundant but interdependent role for both groups, suggesting a partial overlap but subfunctionalization among class E genes. Furthermore, characterization of *AGL6* clade mutants of rice and maize indicated a similar functional role in floral meristem determinacy and organ identity [114,115,159]. Similar expression patterns of monocot *AGL6* and *SEP* clade genes and their complex interactions with class B, C, and D genes indicated conserved floral specification systems. The functional similarity of *SEP* and *AGL6* clades is provided by Petunia floral development genes [173].

## **6. Future Perspectives**

Understanding of grass inflorescence morphogenesis has expanded significantly over the last two decades. Extensive studies in model plants have demonstrated common genetic factors regulating eudicot and grass floral development including MADS-box and non-MADS-box genes and epigenetic regulators. For Arabidopsis and rice, the genetic and molecular mechanisms of transition from vegetative to reproductive phase and the role of MADS-box genes in floral organ identity are well understood. However, this is less well defined for Brachypodium, maize, and wheat because loss-of-function mutant analysis is rare in these species. Currently, advances in genetics analysis has made mutant development and characterization easy in grasses which is being used to define grass floral developmental biology. Deciphering the molecular control of transition from shoot apical meristem to floral meristem development and the determination of floral organ identity will provide new insights to devise innovative strategies for the development of cereals with enhanced grain yield and adaptation to multiple environments [174].

Biological research in general and plant evolutionary biology have been revolutionized by advances in next generation sequencing. Enormous amounts of genomic and transcriptomic sequence data have also been generated through the 1000 Plants Project (1KP) and the Floral Genome Project [175,176]. These gene resources provide an unprecedented opportunity to bridge the evolutionary gap between floral morphogenesis in model plants and economically important cereals by characterizing floral genetic components of ABCD model. Here, it is important to note that most of the cereal orthologs are merely retrieved by phylogenetic analysis from resource genome databases; therefore, specified experimental studies will be required to support genetic framework of underlying mechanisms of floral organ identity in cereals. In this perspective, genome editing tools such as virus-induced gene silencing (VIGS) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been widely useful in several eudicots and monocot species to investigate gene function in floral organ identity and symmetry between basal and core eudicots [177–181]. Effective application of these systems could herald a new generation of multidisciplinary evo–devo research that better describes the evolutionary changes in gene regulatory networks underlying floral development. Moreover, these approaches along with TILLING resources can provide new avenues for grain yield improvement in cereals through translational research.

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/11/ 2743/s1.

**Author Contributions:** Z.A. conceived the idea. Z.A., Q.R., and R.M.A. retrieved relevant literature and drafted the manuscript. Q.R., R.M.A., and M.A. performed phylogenetic analysis, interpreted results, and drew figures. Z.A., U.A., and G.C. read, reviewed, and edited the manuscript. All authors listed have made substantial, direct, and intellectual contribution to the work and approved the final manuscript.

**Funding:** This work was carried out with the support of "Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ014344)" Rural Development Administration, Korea.

**Acknowledgments:** The authors gratefully acknowledge the review effort of Richard Trethowan (University of Sydney, Australia) for improving the manuscript and financial support from the USPCAS-AFS, University of Agriculture Faisalabad, Pakistan and Higher Education Commission of Pakistan.

**Conflicts of Interest:** The authors declare no conflicts of interest.

## **Abbreviations**



