Identification of plant enhancers and their constituent elements by STARR-seq in tobacco leaves

Abstract

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 ...

Synthetic Promoter Designs Enabled by a Comprehensive Analysis of Plant Core Promoters

“…Consistent with previous reports (Bruce et al, 1989;Jores et al, 2020), the 35S enhancer was four-fold more active in the tobacco system than in maize protoplasts (Fig. 1b).…”

“…TF presence and activity cannot simply be inferred from TF motifs because plant TF families are large and often encode both activating and repressing factors with highly similar binding preferences. However, single cell genomics can determine which TFs are expressed in specific cell types and associated with chromatin accessibility of regulatory elements (Dorrity et al, 2020;Marand et al, 2020). This knowledge offers a promising avenue to explore the activity of cell type-specific regulatory elements.…”

“…The plant STARR-seq assay can identify light-responsive enhancers (Jores et al, 2020). To test whether core promoters that respond to light can also be identified, we subjected the promoter libraries to STARR-seq experiments in tobacco leaves that were kept in the light (16h light, 8h dark) for two days after transformation ( Fig.…”

“…However, it is not clear whether these rules also apply to plant promoters. Although computational analyses have revealed that many of the core promoter elements identified in animals are enriched in plant promoters (Molina and Grotewold, 2005;Yamamoto et al, 2007;Kumari and Ware, 2013), only the TATA-box and the Initiator (Inr) element have been functionally validated (Zhu et al, 1995;Kiran et al, 2006;Srivastava et al, 2014;Jores et al, 2020). Some plant promoters do not harbor any of the known core promoter elements (Kumari and Ware, 2013).…”

“…12 | Light-responsive plant enhancers are not active in maize protoplasts. Constructs harboring no enhancer (none), a 35S enhancer, or one of three light-responsive plant enhancers (AB80, Cab-1, or rbcS-E9) upstream of the 35S minimal promoter were subjected to STARR-seq in maize protoplasts generated from dark-grown plants (Jores et al, 2020). Each boxplot (center line, median; box limits, upper and lower quartiles; whiskers, 1.5 × interquartile range; points, outliers) denotes the enrichment of all recovered mRNA barcodes over the DNA input.…”