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Title: 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

Author
item WEN, ZHIFENG - Northwest Agricultural & Forestry University
item YANG, YAZHOU - Northwest Agricultural & Forestry University
item ZHANG, JINJIN - Shaanxi Normal University
item WANG, XIPING - Northwest Agricultural & Forestry University
item LIU, ZHONGCHI - University Of Maryland
item SINGER, STACY - University Of Alberta
item YANG, YINGJUN - Henan Agricultural University
item Liu, Zongrang

Submitted to: Plant Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/22/2014
Publication Date: 6/4/2014
Citation: Wen, Z., Yang, Y., Zhang, J., Wang, X., Liu, Z., Singer, S., Yang, Y., Liu, Z. 2014. 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. Plant Biotechnology. DOI: 10.1111/pbi.12203.

Interpretive Summary: Genetic engineering improves desirable traits in plants using a gene that is controlled by a specific promoter. Introducing multiple genes into a plant requires multiple promoters. We hypothesized that multiple promoters could interact and alter both the gene action and other promoters. We devised a new approach to study promoter-promoter interactions and found that the promoters do interact resulting in unexpected gene expression and gene performance. This work is the first of its kind to demonstrate that there are potential problems with the use of multiple tissue-specific gene-promoters within a plant transformation vector and calls attention to the need for technologies to prevent interaction in multiple gene systems in order to achieve the expected gene performance.

Technical Abstract: Molecular stacking via a single transgene enables multiple traits being engineered efficiently in crops. However, whether distinct plant promoters co-existed in the same transgene could, like their mammalian counterparts, mutually interact or interfere remains unknown. In this study, researchers devised a novel approach to investigate the enhancer- and promoter-promoter interactions in transgenic plants. This approach demonstrated that three of four flower-specific enhancer/promoters tested, even located at 2.5 kb apart, were capable of activating the pollen- and stigma-specific Pps promoter driving a cytotoxic DT-A in the tissues other than pollen and stigma, as reflected by tissue-specific DT-A up-regulation and ablation. Researchers showed that the NtAGI1 enhancer activated petal-, stamen- and carpel-specific DT-A expression and ablation in an orientation-independent manner, and this activation was completely abolished by the insertion of an enhancer-blocking insulator, EXOB, between them. This indicated that NtAGI1 directly acts on the Pps promoter rather than DT-A. AGL8 and AP1Lb1 but not AP1La promoters similarly activated distinct tissue-specific DT-A expression and ablation patterns, with the former inciting global growth retardation while the latter ablating the apical inflorescences. The enhancer/promoters’ specificity appears to dictate their tissue-specific activation action but in a strength-/activity-dependent manner. Our work presents the first evidence that plant-derived, flower-specific enhancer/promoters can distantly interact in transgene, which could pose a potential problem for tissue-specific engineering of multiple traits using gene-link approach. This entails taking necessary measures (e.g. use of genetic insulators) to prevent such interaction in molecular stacking gene constructs. Practical and fundamental significance of this work will be discussed.