Induced expression of AtLEC1 and AtLEC2 differentially promotes somatic embryogenesis in transgenic tobacco plants

Abstract

Arabidopsis LEAFY COTYLEDON (LEC) genes, AtLEC1 and AtLEC2, are important embryonic regulators that play key roles in morphogenesis and maturation phases during embryo development. Ectopic expression of AtLEC1 and AtLEC2 in tobacco caused abnormality in transgenic seedling. When transgenic seeds germinated on medium containing 30 µM DEX, LEC1 transgenic seedlings were ivory and fleshy, with unexpanded cotyledons, stubby hypocotyls, short roots and no obvious callus formation at the shoot meristem position. While LEC2 transgenic seedlings formed embryonic callus on the shoot apical meristem and somatic embryo-like structures emerged from the surface of the callus. When callus were transferred to hormone free MS0 medium more shoots were regenerated from each callus. However, shoot formation was not observed in LEC1 overexpressors. To investigate the mechanisms of LEC2 in somatic embryogenesis, we studied global gene expression by digital gene expression profiling analysis. The results indicated that ectopic expression of LEC2 genes induced accumulation of embryo-specific proteins such as seed storage proteins, late embryogenesis abundant (LEA) proteins, fatty acid biosynthetic enzymes, products of steroid biosynthesis related genes and key regulatory genes of the embryo development. Genes of plant-specific transcription factors such as NAC domain protein, AP2 and GRAS family, resistance-related as well as salicylic acid signaling related genes were up-regulated in LEC2 transgenic seedlings. Ectopi c expression of LEC2 induced large number of somatic embryo formation and shoot regeneration but 20 d DEX induction of LEC1 is not sufficient to induce somatic embryogenesis and shoot formation. Our data provide new information to understand the mechanisms on LEC2 gene's induction of somatic embryogenesis.

Figure 1. Hypocotyl and root length of transgenic and WT tobacco germinated on 30 µM DEX containing medium for 20 d.

The LEC1 transgenic seedling roots were shorter, but the LEC2’s were longer than the control.

Figure 2. LEC1 transgenic and WT tobacco seeds germinated on 30 µM DEX medium.

(A, B): comparison of LEC1 transgenic and WT tobacco seedlings on DEX medium for 12 d. (C) LEC1 transgenic seedlings grown on 30 µM DEX medium for 25 d.

Figure 3. LEC2 transgenic and WT tobacco seeds germinated on 30 µM DEX medium.

(A, B) LEC2 transgenic and wild type seedlings grown on 30 µM DEX medium for 20 d. (C) the close view of the callus in A. (D) shoot formation from callus of LEC2 transgenic seedling grown on 30 µM DEX medium for 30 d.