Abstract:

Flavonols play important roles in plant responses to various environmental stresses and in the regulation of seed coat development. In this study， a transparent seed coat mutant was obtained through ethyl methane sulfonate（EMS） mutagenesis screening， compared with wild-type Arabidopsis thaliana（Col-0）， the mature seed color of the mutant was yellow， and its phenotypic trait was controlled by a recessive single gene. Using map-based cloning and fine-mapping technology， the mutant gene TT4（At5G13930） was mapped to BAC of MAH20 on chromosome 5， and the 1 299th base C of the TT4 was mutated to T， resulting in the 324th amino acid glycine mutated to glutamic acid. TT4（transparent testa 4） encoded a structural gene for flavonoid synthesis， chalcone synthase（CHS）， and after mutation， the seed coat was transparent and the seed color was yellow， then the mutant was named tt4-1. The phenotype of brown seed coat was restored by functional complement mutants， which further demonstrated the important role of TT4 in regulating the development of seed coat color. The tissue expression analysis of TT4 was conducted by promoter coupling GUS assay， and it was found that TT4 gene was expressed in the roots， stems， leaves of plant seedlings and flowers respectively. Physiological phenotypic analysis showed that the mutant tt4-1 seeds germinated earlier， and the seedlings had shorter taproots， as well as more lateral roots and root hairs， larger stomatal opening and higher water loss rate in mature leaves compared with the wild type. This study would lay a theoretical basis for further elaborating the function of TT4 gene.

Key words: Transparent testa 4, screening, flavonoids, map-based cloning