PNNL

Abstract

As plant synthetic biology is rapidly leading to large-scale production of secondary metabolites and agronomic products, synthetic promoters have emerged as a useful component to construct stress induced genes in crops. Synthetic promoters are rationally designed using short cis-regulatory elements (CREs) and core promoter sequences. Since CREs are fundamental to effective and specific gene induction in diverse plant systems, reliable CRE selection and arrangement in synthetic promoters are essential factors to consider during design. We identified novel conserved DNA motifs from the promoter sequences of genes differentially expressed during water deficit stress in leaf palisade and vascular cell types of hybrid poplar (Populus tremula * P. alba) isolated using laser capture microdissection technique. Based on these motifs, four sequences were hexamerized (6 × 20 bp) and inserted into synthetic promoters for reporter gene assays. Two of these synthetic promoters (Syn2 and Syn3) induced GFP in transformed poplar mesophyll protoplasts in 0.5 M mannitol incubation. Two shorter synthetic promoters (6 × 10 bp), Syn3-10b-1 (5’) and Syn3-10b-2 (3’), were generated from the Syn3 basic sequence. Syn3 and Syn3-10b-1 were specifically induced in transient agroinfiltration in Nicotiana benthamiana leaves under water-deficit conditions. In transgenic poplar, only one of these promoters (Syn3-10b-1) was induced in green tissues under water-deficit stress conditions while a very low induction and expression was observed in roots. Syn3-10b-2 did not induce water-deficit specific expression in any tissues, which was consistent with transient transformation data. Interestingly, Syn3 induced consistent GUS expression in all tissues with or without water-deficit stress, but expression was much higher in green tissues, especially leaves. Therefore, a synthetic promoter containing the 5’ sequence of Syn3 endowed both tissue-specificity and water-deficit inducibility in transgenic poplar, whereas the 3’ sequence did not. This study adds two new synthetic promoters to the poplar engineering toolkit: Syn3-10b-1, which is a green tissue-specific and water stress induced promoter, and Syn3, which is a green tissue preferred constitutive promoter.