AtDEAHb3, A DEAH-box RNA Helicase, Involved In Arabidopsis Female Gametophytic Development

Female gametophyte plays a critical role in the sexual reproduction of angiosperms. Successful development of female gametophytes in higher plants is the consequence of coordination of cell specification, proliferation, and differentiation. Several mutants and genes that are involved in female gametophyte development were identified.RNA helicases are involved in mRNA transcription, pre-RNA splicing, RNA maturation, transportation, translation initiation and ribosomal assembly, and many other cellular processes, which play an important role in eukaryotic organism development. Over100RNA helicase genes in Arabidopsis genome were found, but only few of them are being functional analyzed. Previous studies revealed that RNA helicases were involved in regulating plant growth, development and stress responses.In our study, two T-DNA insertion lines were identified and no homozygous insertion was found in their selfed progeny. In the self-pollinated progeny of the heterozygotic mutant plants, the segregation ratio of heterozygous to wild type plants was about1:1, instead of the typical3:1. Reciprocal cross analysis revealed that the mutant allei could not be transmitted through the female gametophyte. There are about50%seeds in the silique of the mutant compared with the wild type. These results indicate that the mutation has a vital effect on the female gametophyte development. The T-DNA was inserted in a DEAH-box RNA helicase gene. Arabidopsis genome codes22DEAH-box RNA helicases. We named this gene as AtDEAHb3.In flower stage12, the majorities of wild-type embryo sacs are mature and develop to the stage FG6or FG7. However, about50%female gametophytes from atdeahb3/+plants show delayed development, from FG2to FG7stages, indicating that the synchrony of female gametophytic development is impaired in atdeahb3pistils.GUS staining analysis in the pAtDEAHb3::GUS transgenic Arabidopsis plants revealed that AtDEAHb3express in the various tissues, especially in the apical meristem and ovule. The expression of AtDEAHb3was detected in the whole process of female gametophytic development by using RNA in situ hybridization.Arabidopsis GFA1is the core component of U5SnRNP complex involved in pre-mRNA splicing. It was predicated that GFA1and AtDEAHb3have similar expression patterns in Arahidopsis development. Furthermore, yeast two-hybrid, BiFC and Pull-Down analysis revealed that AtDEAHb3could physically interact with GFA1, indicating that AtDEAHb3may play a role in female gametophyte development by involving in pre-mRNA splicing.In the AtDEAHb3-amiRNAi transgenic Arabidopsis lines, the expressions of CYCB genes were down-regulated, which indicates that AtDEAHb3functions in the mitotic process by regulating CYCB genes expression and pre-mRNA splicing.