Class C MADS-box gene AGAMOUS was duplicated in the wheat genome

Class C MADS-box gene AGAMOUS was duplicated in the wheat genome

Chizuru Hirabayashi and Koji Murai

Department of Bioscience, Fukui Prefectural University, Eiheiji-cho, Fukui 910-1195, Japan

Corresponding author: Koji Murai

Abstract

Class C MADS-box gene is involved in specifying stamen and carpel identity during flower development in plant species. To obtain information about the molecular mechanism underlying floral organ formation in wheat, we identified two AGAMOUS (AG)-like MADS box genes, WAG-1 (wheat AGAMOUS-1) and WAG-2. Phylogenetic analysis of WAG-1 and WAG-2, together with class C MADS-box genes in barely, rice and maize, indicated that the monocot class C genes are classified into two clades, WAG-1 clade and WAG-2 clade. Arabidopsis AG is more close to WAG-2 clade than WAG-1 clade, suggesting that the genes in the former clade, wheat WAG-2, barley HvAG1, rice OsMADS3, and maize ZMM2, have more similar function to AG.

Flower development has been the subject of intensive study over the last decade, particularly in two dicot species, Arabidopsis thaliana and Antirrhinum majus (Jack 2004). These studies have provided a general understanding of the development of floral organs in higher plants and led to the production of the ABCDE model. This model postulates that floral organ identity is defined by five classes of homeotic gene, named A, B, C, D and E (Zahn et al. 2006). According to the ABCDE model, class A and E genes specify sepals in the first floral whorl, class A, B and E genes specify petals in the second whorl, class B, C and E genes specify stamens in the third whorl, class C and E genes specify carpels in the fourth whorl, and class D and E genes specify the ovule in the pistil. Cloning of ABCDE organ identity genes in Arabidopsis showed that they encode MADS-box transcription factors except for the class A gene, APETALA2 (AP2). The class A MADS-box gene is AP1, the class B genes are AP3 and PISTILLATA (PI), the class C gene is AGAMOUS (AG), and the class D gene is SEEDSTICK (STK). In Arabidopsis, the class E genes consist of four members, SEPALLATA1 (SEP1), SEP2, SEP3 and SEP4, which show partially redundant functions in identity determination of petals, stamens and carpels.

Analysis of the ABCDE genes in monocot species such as rice suggests that the ABCDE model could essentially be extended to monocots except for the role of the class A gene (Kater et al. 2006; Yamaguchi and Hirano 2006). In wheat, it has been reported that the AP1-like gene WAP1 (wheat AP1, sometimes called VRN1) (Murai et al. 1998; Murai et al. 2002) has no class A function but acts in phase transition from vegetative to reproductive growth (Murai et al. 2003). Using alloplasmic wheat line which shows pistillody, homeotic transformation of stamens into pistil-like structures, we identified a wheat AP3 ortholog, WAP3 (wheat APETALA3) (Murai et al. 1998), and two wheat PI orthologs, WPI-1 (wheat PISTILLATA-1) and WPI-2 (Hama et al. 2004) as the wheat class B genes. Furthermore, we identified two types of class E genes from wheat, WSEP (wheat SEPALLATA) and WLHS1 (wheat Leafy Hull Sterile 1) (Shitsukawa et al. 2007). Our functional analysis indicated that WSEP is more likely to be orthologs of class E genes than WLHS1. As class C gene, we previously identified WAG-1 (wheat AG-1) (Meguro et al. 2003), but its function is unclear. Here we report the second wheat AG-like gene, WAG-2.

WAG-2 was isolated from a wheat expressed sequence tag (EST) database (Ogihara et al. 2003). By screening all the EST contigs through a BLAST search, we identified a contig with high sequence similarity to OsMADS3 in rice (Kang et al. 1995) and ZMM2 in maize (Theissen et al. 1995), and named WAG-2. To inspect the relationship between wheat AG-like genes, WAG-1 and WAG-2, and other members of the class C gene family in detail, the phylogenetic tree was reconstructed by using the amino acid sequences (Fig. 1). The phylogenetic tree indicated that the monocot class C gene family separated into two groups, WAG-1 clade and WAG-2 clade. The WAG-1 clade contains barley HvAG2, rice OsMADS58 (Yamaguchi et al. 2006) and maize ZAG1 (Mena et al. 1996), whereas the WAG-2 clade insists of barley HvAG1, rice OsMADS3 (Kang et al. 1995) and maize ZMM2 (Theissen et al. 1995). This indicates that AG orthologs were duplicated in each monocot species. The phylogenetic tree also indicates that dicot class C genes, Arabidopsis AG and Antirrhinum PLE, are more close to WAG-2 clade than WAG-1 clade (Fig. 1). This suggests that the genes in the former clade, wheat WAG-2, barley HvAG1, rice OsMADS3, and maize ZMM2, have more similar function to dicot class C genes. Functional analyses were performed in rice AG orthologs by using mutant and RNAi transgenic plants (Yamaguchi et al. 2006). The mutant and transgenic studies indicated that OsMADS3 plays a more predominant role in inhibiting lodicule development and in specifying stamen identity, whereas OsMADS58 contributes more to conferring floral meristem determinacy and to regulating carpel morphogenesis. It means that the duplicated class C genes in rice, OsMADS3 and OsMADS58, show only partial conservation of function with the Arabidopsis class C gene, AG. Interestingly, carpel identity is determined by a YABBY gene named DROOPING LEAF (DL) in rice (Nagasawa et al., 2003; Yamaguchi et al., 2004).

Wheat (Triticum aestivum L.) is a hexaploid species with the genome constitution AABBDD that originated from three diploid ancestral species: the A genome came from T. urartu, the B genome from Aegilops speltoides or another species classified in the Sitopsis section, and the D genome from Ae. tauschii (Feldman 2001). Allopolyploidization leads to the generation of duplicated homoeologous genes (homoeologs), as opposed to paralogous genes (paralogs). Consequently, the hexaploid wheat genome contains triplicated homoeologs derived from the ancestral diploid species. In the wheat EST database, we can find four other AG-like genes in addition to WAG-1 and WAG-2. Our phylogenetic analysis indicated that three genes, WM29A, WM29B, and AGL39, are classified into the WAG-2 clade (Fig. 1). WM29A (identical with TaAG-2A) and WM29B (identical with TaAG-2B) were identified from cv. Chinese Spring (Paolacci et al. 2007), and AGL39 was from cv. Nongda 3338 (Zhao et al. 2006). WAG-2, WM29A and WM29B should be three homoeologs in the A, B, or D genome of Chinese Spring wheat. Our phylogenetic analysis also indicated that WAG-1 makes subclade with WM2 (TaAG-1) (Fig. 1). WAG-1 was identified from cv. Norin 26 (Meguro et al. 2003), but WM2 was from cv. Chinese Spring (Paolacci et al. 2007).

In conclusion, the wheat genome contains two AG orthologs, WAG-1 and WAG-2, and at least in WAG-2, there are three homoeologs located in the A, B, or D genomes. Functional diversification of three homoeologs should be examined in the future work.

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