Bread wheat (Triticum aestivum) is a hexaploid species with A, B, and D ancestral genomes. Most bread wheat genes are present in the genome as triplicated homoeologous genes (homoeologs) derived from the ancestral species. Here, we report that both genetic and epigenetic alterations have occurred in the homoeologs of a wheat class E MADS box gene. Two class E genes are identified in wheat, wheat SEPALLATA (WSEP) and wheat LEAFY HULL STERILE1 (WLHS1), which are homologs of Os MADS45 and Os MADS1 in rice (Oryza sativa), respectively. The three wheat homoeologs of WSEP showed similar genomic structures and expression profiles. By contrast, the three homoeologs of WLHS1 showed genetic and epigenetic alterations. The A genome WLHS1 homoeolog (WLHS1-A) had a structural alteration that contained a large novel sequence in place of the K domain sequence. A yeast two-hybrid analysis and a transgenic experiment indicated that the WLHS1-A protein had no apparent function. The B and D genome homoeologs, WLHS1-B and WLHS1-D, respectively, had an intact MADS box gene structure, but WLHS1-B was predominantly silenced by cytosine methylation. Consequently, of the three WLHS1 homoeologs, only WLHS1-D functions in hexaploid wheat. This is a situation where three homoeologs are differentially regulated by genetic and epigenetic mechanisms.

Homeotic transformation of stamens into pistil-like structures (pistillody) has been reported in cytoplasmic substitution (alloplasmic) lines of bread wheat (Triticum aestivum L.) that have the cytoplasm of a related wild species, Aegilops crassa. An ectopic ovule differentiates in the pistil-like stamen in the alloplasmic wheat. The SEEDSTICK (STK)-like class D MADS-box gene, wheat STK (WSTK), was expressed in the primordia of ectopic ovules in the pistil-like stamens as well as in the true pistil, suggesting that ectopic ovule formation results from WSTK expression in the pistil-like stamens of alloplasmic wheat. The ectopic ovule is abnormal as it fails to form complete integuments. Based on the expression pattern of WSTK and B(sister) MADS-box gene, WBsis (wheat B ( sister )), we conclude that WSTK plays a role in determination of ovule identity in the pistil-like stamen, but complete ovule development fails due to aberrant expression of WBsis.

The DROOPING LEAF (DL) gene regulates carpel specification in the flower and midrib formation in the leaf in Oryza sativa (rice). Loss-of-function mutations in the dl locus cause homeotic transformation of carpels into stamens and lack of midrib, resulting in the drooping leaf phenotype. DL is a member of the YABBY gene family and is closely related to the CRABS CLAW (CRC) gene in Arabidopsis thaliana. The function of Arabidopsis CRC, however, differs from that of rice DL: it is responsible for nectary development and is partially involved in carpel identity. Thus, genes related to DL/CRC seem to have functionally diversified during angiosperm evolution. To assess the conservation of DL function in related species, here we examined the in situ expression patterns of DL orthologs in three grass species, i.e., maize, wheat and sorghum, which is assigned to subfamilies different from Ehrhartoideae including O. sativa. The results clearly show that the temporal and spatial expression patterns of DL orthologs in the three species are identical to those of rice DL in both flower and leaf development, suggesting that DL-related genes are functionally conserved within the grass family. It is likely that DL may have been recruited to carpel specification and midrib formation within the lineage of the grass family after divergence of their ancestor from that of eudicots.

Homeotic transformation of stamens into pistil-like structures, called pistillody, has been reported in some alloplasmic common wheat lines with Aegilops crassa cytoplasm. An alloplasmic line of Chinese Spring ditelosomic 7BS (CSdt7BS) with Ae. crassa cytoplasm lacking the long arm of the chromosome 7B shows pistillody, and the pistils and transformed stamens are sterile due to abnormal ovule development. To elucidate the molecular mechanism of the ovule abnormality, we compared the expression profiles of floral organs between euplasmic and alloplasmic CSdt7BS lines. Two differential display methods of mRNA profiling demonstrated that Ae. crassa cytoplasm largely affects nuclear gene expression profiles of common wheat. Of the differentially expressed genes, a wheat AINTEGUMENTA (ANT) homolog, WANT-1, was preferentially expressed in pistils but not in stamens, and accumulation of the transcript was limited to ovule primordia at the floral organ development stage. In alloplasmic wheat, WANT-1 expression was patchy and weak at the ovule-development stages. On the other hand, no significant difference in gene expression patterns of wheat AGAMOUS (AG) homologs (WAG-1 and WAG-2) was observed between fertile and sterile pistils. These results indicated that alteration of gene expression after initiation of ovule primordia results in abnormal ovule development, and that the aberrant ovule formation is at least partly associated with the weak expression of WANT-1 around ovule primordia in alloplasmic wheat with Ae. crassa cytoplasm.

BackgroundAlloplasmic wheat lines with Aegilops crassa cytoplasm often show homeotic conversion of stamens into pistils under long-day conditions. In the pistillody-exhibiting florets, an ectopic ovule is formed within the transformed stamens, and female sterility is also observed because of abnormal integument development.ResultsIn this study, four wheat Bell1-like homeobox (BLH) genes were isolated and named WBLH1 to WBLH4. WBLH1/WBLH3/WBLH4 expression was observed in the basal boundary region of the ovary in both normal pistils and transformed stamens. WBLH2 was also strongly expressed in integuments not only of normal ovules in pistils but also of the ectopic ovules in transformed stamens, and the WBLH2 expression pattern in the sterile pistils seemed to be identical to that in normal ovules of fertile pistils. In addition, WBLH1 and WBLH3 showed interactions with the three wheat KNOX proteins through the BEL domain. WBLH2, however, formed a complex with wheat KNOTTED1 and ROUGH SHEATH1 orthologs through SKY and BEL domains, but not with a wheat LIGULELESS4 ortholog.ConclusionsExpression of the four WBLH genes is evident in reproductive organs including pistils and transformed stamens and is independent from female sterility in alloplasmic wheat lines with Ae. crassa cytoplasm. KNOX-BLH interaction was conserved among various plant species, indicating the significance of KNOX-BLH complex formation in wheat developmental processes. The functional features of WBLH2 are likely to be distinct from other BLH gene functions in wheat development.