Cloning, Characterization, and Functional Analysis of Seed Coat Mucilage-related Gene TTG1 from Lepidium perfoliatum

Lepidium perfoliatum, an annual herb plant species of Brassicaceae, has a typical myxospermy. The TTG1 (Transparent testa glabra 1) gene encodes a putative transcription factor, which has been identified to play a role in epidermal cell differentiation and mucilage release. Till now, research on the TTG1 gene in myxospermy plants has been rarely reported. To identify TTG1 gene function, mucilage related gene LpTTG1 from L. perfoliatum was cloned in the present study. The full length ORF of the TTG1 gene from L. perfoliatum was isolated by homologous cloning, and was found to be 1032 bp long, encoded 343 putative amino acids and contained WD40 motifs, named as LpTTG1. The qRT-PCR results showed that LpTTG1 was widely expressed in different tissues of L. perfoliatum, which may reflect diverse functions of LpTTG1. Moreover, immunolocalization analysis indicated that the expression of LpTTG1 changed in inner and outer integuments and corresponded to the synthesis of mucilage in outer integument cell layers, suggesting that LpTTG1 mainly regulates the development of the seed coat then applies the effect on mucilage production. Furthermore, overexpression of LpTTG1 in Arabidopsis could significantly enhance the expression of AtMUM4 (which developmentally regulates mucilage production downstream) in silique, which means LpTTG1 attends to the mucilage regulation pathway and generates more downstream product-MUM4 in promotion of mucilage synthesis. In our experiment, however, no significant difference in seed morphology and release pattern, or the secretion amount of mucilage between LpTTG1 overexpression transgenic line and WT, was observed. One possible explanation may be that mucilage synthesis and release is a complex process in Arabidopsis, and is regulated by many genes with functional redundancy, therefore increasing the transcription level with one of them during development may not result in significant phenotype change.