Targeted engineering of plant gene expression holds great promise for ensuring food security and for producing biopharmaceuticals in plants. However, this engineering requires thorough knowledge of cis-regulatory elements in order to precisely control either endogenous or introduced genes. To generate this knowledge, we used a massively parallel reporter assay to measure the activity of nearly complete sets of promoters from Arabidopsis, maize and sorghum. We demonstrate that core promoter elements -notably the TATA-box -as well as promoter GC content and promoterproximal transcription factor binding sites influence promoter strength. By performing the experiments in two assay systems, leaves of the dicot tobacco and protoplasts of the monocot maize, we detect species-specific differences in the contributions of GC content and transcription factors to promoter strength. Using these observations, we built computational models to predict promoter strength in both assay systems, allowing us to design highly active promoters comparable in activity to the viral 35S minimal promoter. Our results establish a promising experimental approach to optimize native promoter elements and generate synthetic ones with desirable features.

A high-throughput assay in transiently transformed tobacco leaves identifies enhancers, characterizes their functional elements and detects condition-specific enhancer activity.

In a time of climate change and increasing human population, crop plants with higher yields and improved response to abiotic stresses will be required to ensure food security. As many of the beneficial traits in domesticated crops are caused by mutations in cis-regulatory elements, especially enhancers, genetic engineering of such elements is a promising strategy for improving crops 1,2 . However, such engineering is currently hindered by our limited knowledge of plant cis-regulatory elements. Here, we adapted STARRseq -a technology for the high-throughput identification of enhancers -for its use in transiently transfected tobacco leaves. We demonstrate that the optimal placement in the reporter construct of enhancer sequences from a plant virus, pea and wheat was just upstream of a minimal promoter, and that none of these four known enhancers was active in the 3′-UTR of the reporter gene. The optimized assay sensitively identified small DNA regions containing each of the four enhancers, including two whose activity was stimulated by light. Furthermore, we coupled the assay to saturation mutagenesis to pinpoint functional regions within an enhancer, which we recombined to create synthetic enhancers. Our results describe an approach to define enhancer properties that can be performed in any plant species or tissue transformable by Agrobacterium and that can use regulatory DNA derived from any plant. MainEnhancers can be identified by self-transcribing active regulatory region sequencing (STARR-seq), a massively parallel reporter assay 3 . Here, candidate enhancer sequences are inserted into the 3' untranslated region (3′-UTR) of a reporter gene under the control of a minimal promoter. If an insert has enhancer activity, it can upregulate its own transcription. The resulting transcript can be detected by next generation sequencing and linked to its corresponding enhancer element, which is incorporated within the mRNA. This method has been widely used in Drosophila and human cells. In plants, STARR-seq has been described in only two studies that apply the method to the monocot species rice and maize 4,5 . Both studies relied on species-specific protoplasts as recipient cells for the assay. Our approach bypasses the need for a species-specific protoplasting protocol by using transient expression of diverse STARR-seq libraries in tobacco leaves. As transcription factors are highly conserved among plant species 6,7 , the versatile tobacco system can serve as a proxy for many plant species, including crops.We used a green fluorescent protein (GFP) reporter gene under control of the Cauliflower mosaic virus 35S minimal promoter and the 35S core enhancer 8,9 (subdomains A1 and B1-3) to benchmark the assay.We systematically analyzed the position-and orientation-dependency of the enhancer (Fig. 1a). To this end, we used a more generalized version of STARR-seq in which we placed a barcode in the GFP open reading frame. This barcode is linked to the corresponding enhancer variant by next generation sequencing and serves as a ...

Targeted engineering of plant gene expression holds great promise for ensuring food security and for producing biopharmaceuticals in plants. However, this engineering requires thorough knowledge of cis-regulatory elements in order to precisely control either endogenous or introduced genes. To generate this knowledge, we used a massively parallel reporter assay to measure the activity of nearly complete sets of promoters from Arabidopsis, maize and sorghum. We demonstrate that core promoter elements – notably the TATA-box – as well as promoter GC content and promoter-proximal transcription factor binding sites influence promoter strength. By performing the experiments in two assay systems, leaves of the dicot tobacco and protoplasts of the monocot maize, we detected species-specific differences in the contributions of GC content and transcription factors to promoter strength. Using these observations, we built computational models to predict promoter strength in both assay systems, allowing us to design highly active promoters comparable in activity to the viral 35S promoter. Our results establish a promising experimental approach to optimize native promoter elements and generate synthetic ones with desirable features.