Isolation Of Floral Organ Identity Genes And Hormonal Regulation Of Their Expression In Hyacinthus Orientalis L.

Floral organs have been successfully induced from the regenerated floral buds of ffyacinthus orienralis L. by precisely controlling exogenous hormones in the medium. Under high concentration of cytokinin and auxin, the regenerated floral bud produces only tepals. However, at reduced levels of the hormones, the regenerated floral bud can produce stamens and/or carpels with ovules. This provides a very ideal system to understand the molecualr mechanism of hormone-regualted flower development. In this study, we isolated floral identity genes HAG1, HPI1 and HAP2 and determined their expressions both in vivo and in vitro. Main results and conclusions are as follows:1 HAG1 cDNA is 1061bp in full length, and it encodes a putative polypeptide of 229 amino acids. HAG1 lacks N-terminal extension domain, and has the conserved MADS-box. Its amino acid sequence is 80% homologous to AG in Arabidopsis.2 Hybridization results show that HAG1 is specifically expressed in stamens and carpels in vivo; HAG1 mRNA was first detected in cultured explants at day 5 in the medium containing high levels of cytokinin and auxin,which could induce floral regeneration in vitro. However, no HAG1 mRNA was detected in the cultured explants until day 10 in media with low or no hormones. Further, HAGJ mRNA was detected in the stamens and carpels of regenerated floral buds, but not in the tepals.3 To understand HAG1 function, we transformed it to Arabidopsis plant. A transcriptional fusion between 35S promoter and nearly full length HAG1 cDNA was constructed and transformation was performed through agrobacterium using rootexpantSo The first whorl organs of transgenic plants flowers are carpel-like organs with stigmatic papilllae and a few ovules. The second whorl organs in these transformants usually have staminoid petals or stamens or are petalless. The third and fourth whorl organs are normal.4 HPH is also a MADS-box gene, the full length cDNA is 888bp. It encodes a putative protein with 210 amino acids. Sequence comparison analysis reveales that HPI1 has a high degree of nucleotide sequence similarity to that of PI in Arabidopsis. However, different from PI, HPI1 is expressed not only in tepals and stamens, but also in carpels. HPH mRNA is also detected in regenerated floral buds and its stamens.5 The size ofHAP2 cDNA is 1697bp, and its encoding fragment composes of 368 amino acids. Similar to AP2 in Arabidopsis, HAP2 have a highly basic 10-amino acid domain that includes a putative nuclear located sequence KKSR. HAP2 also contains two copies of conserved repeated domain. The residues capable of forming amphipathic a-helice structure in the first conserved domain are identical to those of AP2, while the structure in the second conserved domain lacks 9 amino acids as compared to that of AP2. RT-PCR combined southern hybridization analysis show that HAP2 is expressed in leaves, tepals, and tepals, stamens of floral buds.6 Analysis of sequences and expression patterns reveals that HAG HPI1 and HAP2 are the homologs ofAG, PI and AP2 in Arabidopsis, respectively. Particularly, overexpression of HAG 1 in Arabidopsis created flower phenotypes resembling that of the apetala2 mutant and AG transgenic Arabidopsis plants. This further confirms that HAG1 is the ortholog of floral identity gene AG. HAG1 expression is activated by hormones, and its activity may be required for the induction of floral bud and the determination of stamen and carpel during the regeneration of floral buds. However, both HPI1 and HAP2 expressions are not induced by exogenous hormones.