Brassica Napus Bna.TTG2 Functions In ABA-mediated Seed Germination

Dormancy and germination are two important characteristics of plant seeds for surviving and adapting to environmental changes. Many kinds of plant hormones are involved in the regulation of seed dormancy and germination process, including ABA. Previous research in our laboratory has shown that the ttg2 mutants in Arabidopsis was not sensitive on exogenous ABA during seed germination, suggested that At TTG2 gene may be involved in the response to ABA during seed germination. However, it is not clear whether the TTG2 gene in Brassica napus, which has high levels of identity with the Arabidopsis TTG2 homologue, is also involved in ABA-mediated seed germination. Here we studied the function of the Bna.TTG2 in seed dormancy and germination, and the regulatory mechanism of ABA responses path way in Arabidopsis. The main results are as follow:(1) We treated the seeds of WT, Bna A.TTG2.a.1-overexpressing and ttg2-3 mutant Arabidopsis with exogenous ABA and surveyed their germination trends. This result indicated that Bna A.TTG2.a.1-overexpressing Arabidopsis was hypersensitive to ABA in seed germination, under the ABA treatment. The germination trends of OE-5-2 was significantly lower than that in wild type, while the germination trends of ttg2-3 is dramatically higher than wild type. It is suggested that Bna A.TTG2.a.1 may be involved in the process of ABA mediate seed germination.(2) We analyzed the differences of seeds dormant degree in WT, Bna A.TTG2.a.1-overexpressing and ttg2-3 mutant Arabidopsis. We found that the seed dormant degree of ttg2-3 mutants was significantly lower than that of wild type; while Bna A.TTG2.a.1-overexpressing Arabidopsis had no significant differences with wild type. In addition, ABA content analysis results showed that the ABA content in the ttg2-3 mutant seeds was significantly lower than that of wild type; and the Bna A.TTG2.a.1-overexpressing Arabidopsis had no obvious difference to wild type. At TTG2 affect the seed dormancy is mainly to the ABA content in seeds. The results show that the At TTG2 regulates seed dormancy mainly concerned with the low content of ABA in seeds.(3) Using a yeast two-hybrid screening, we identified 11 TFs that interact with Bna A.TTG2.a.1. Among them, 7 TFs which had strong X-Gal chromogenic were further confirmed for the interaction with Bna A.TTG2.a.1 by BIFC assays.(4) Through constructing multiple T-DNA insertion lines and dual-overexpression Arabidopsis materials, we analysed the interaction between At TTG2 and TCP14. We obtained tcp14, tcp15, tcp14/ttg2, tcp15/ttg2 and tcp14/tcp15/ttg2 Arabidopsis mutants and ten Bna A.TTG2.a.1 and At TCP14 dual-overexpression T1 lines.(5)To investigated the degree of dormancy and the responses to ABA treatments of these mutant materials, we surveyed the seed germination of WT、ttg2-3、ttg2/tcp14 and tcp14 Arabidopsis after sowing at the filter paper. The experimental result showed that the ttg2-3 mutant dormant degree was significantly lower than wild type; while the dormant degree of ttg2/tcp14 mutant was higher than that of wild type; and tcp14 is the highest. It was suggested that the effect of TCP14 could be greater than At TTG2. Results from treating these materials with ABA showed that the effect of At TTG2 may outweigh the TCP14.