Isolation Of Ovule Identity Gene And Hormonal Regulation Of Its Expression Of Hyacinthus Orientalis L.

Ovule has been as an excellent model system to study morphogenesis and pattern formation of organ. Progress has been made in several aspects of ovule development: the control of ovule identity, the mechanism of early pattern formation, the regulation of integument morphogenesis and embryo sac development. A flowchart outling salient process and known genes involved in the ovule developmental pathway was proposed. However, little is known about the hormonal regulation of ovule initiation and development. In this study, we presented the characterization of a MADS box gene, HoMADS1, from Hyacinthus ovules. Sequence analysis and RNA hybridization revealed that HoMADS1 is most likely a class D MADS box gene. Ectopic expression of the HoMADS1 in Arabidopsis plants promoted the formation of ectopic carpels and ovules. Interestingly, HoMADS1 expression was induced by low levels of cytokinin and auxin which are responsible for the induction of ovule initiation and development in the Hyacinthus regenerated flower. Our data may provide some information to understand the molecular mechanism of ovule development regulated by hormones. The results are as follows:Isolation and characterization of HoMADS1A MADS box gene was isolated from the ovules and was designated as HoMADS1 (Hyacinthus orientalis MADS1). HoMADS1 cDNA is 1,062 bp and containes an open reading frame (ORF) that encodes a deduced protein with 234 amino acid residues. Although sequence comparison showed that HoMADS1 shared less homology with class D gene FBP11 and AGL11 than class C gene CUM10 and HAG1, the phylogenetic analysis of amino acid sequences with class C and D MADS box genes revealed that HoMADS1 is closer to the class D genes, suggesting that HoMADS1 is most likely a class D MADS box gene of Hyacinthus. HoMADS1 is exclusively expressed in the ovulesRNA hybridization analysis showed that HoMADS1 mRNA was detected in the ovule primordia, young ovules and mature ovules. In contrast, no hybridization signals were detected in vegetative organs and other floral organs. This indicates that HoMADS1 is exclusively expressed in the ovules.3. Overexpression of HoMADS1 promotes ectopic carpel and ovule formation in transgenic Arabidopsis plantsWe introduced the sense HoMADS1 under the control of 35S promoter of Cauliflower mosaic virus into Arabidopsis plants for functional analysis. The most marked alteration observed in transgenic plants is that the first whorl organs were completely transformed into carpelloid structures instead of sepals, the stigmatic papillae developed at the tips of the carpelloid structures, and the ovule-like structures formed along the edge of the first whorl organs. Further, longitudinal sections of the ovule-like structures revealed that they contained the female gametophytes, comfirming that these ovule-like structures are real ovules. In addition, it was found that the overexpression of HoMADS1 affected the morphology of cauline leaves close to the inflorescence and they had papillae-like protrusions at their tips. The observation indicated that the leaves were also transformed into the carpelloid structures although they had no ovules. Northern analysis showed that the variation in severity of flower phenotypes of transformants could be caused by the different levels of HoMADS 1 expression.4. In vitro ovule development in HyacinthusThe tepals at later stage of bicellular pollens were used as explants. The regeneration of floral buds and continuous differentiation of tepals were induced by the high levels of cytokinin and auxin in the media. Then, when the hormones were reduced to certain levels, ovules were produced from the regenerated flowers. Microscopic analysis showed that the development of in vitro regenerated ovules resembled that of in planta ovules.5. HoMADS1 is induced by low levels of cytokinin and auxin during in vitro ovule initiation and developmentRNA hybridization was performed to examine the temporal and spatial expression pattern of HoMADS1 in the regenerated floral buds. The results showe...