Highly interactive nature of flower-specific enhancers and promoters, and its potential impact on tissue-specific expression and engineering of multiple genes or agronomic traits.

Abstract

Molecular stacking enables multiple traits to be effectively engineered in crops using a single vector. However, the co-existence of distinct plant promoters in the same transgenic unit might, like their mammalian counterparts, interfere with one another. In this study, we devised a novel approach to investigate enhancer-promoter and promoter-promoter interactions in transgenic plants and demonstrated that three of four flower-specific enhancer/promoters were capable of distantly activating a pollen- and stigma-specific Pps promoter (fused to the cytotoxic DT-A gene) in other tissues, as revealed by novel tissue ablation phenotypes in transgenic plants. The NtAGI1 enhancer exclusively activated stamen- and carpel-specific DT-A expression, thus resulting in tissue ablation in an orientation-independent manner; this activation was completely abolished by the insertion of an enhancer-blocking insulator (EXOB) between the NtAGI1 enhancer and Pps promoter. Similarly, AGL8 and AP1Lb1, but not AP1La, promoters also activated distinct tissue-specific DT-A expression and ablation, with the former causing global growth retardation and the latter ablating apical inflorescences. While the tissue specificity of the enhancer/promoters generally defined their activation specificities, the strength of their activity in particular tissues or developmental stages appeared to determine whether activation actually occurred. Our findings provide the first evidence that plant-derived enhancer/promoters can distantly interact/interfere with one another, which could pose potential problems for the tissue-specific engineering of multiple traits using a single-vector stacking approach. Therefore, our work highlights the importance of adopting enhancer-blocking insulators in transformation vectors to minimize promoter-promoter interactions. The practical and fundamental significance of these findings will be discussed.