Enhancers in Plant Development, Adaptation and Evolution

Abstract

Understanding plant responses to developmental and environmental cues is crucial for studying morphological divergence and local adaptation. Gene expression changes, governed by cis-regulatory modules (CRMs) including enhancers, are a major source of plant phenotypic variation. However, while genome-wide approaches have revealed thousands of putative enhancers in mammals, far fewer have been identified and functionally characterized in plants. This review provides an overview of how enhancers function to control gene regulation, methods to predict DNA sequences that may have enhancer activity, methods utilized to functionally validate enhancers and the current knowledge of enhancers in plants, including how they impact plant development, response to environment and evolutionary adaptation.

Keywords: Adaptation; Enhancer identification; Environmental cues; Functional validation; Plant development; Plant enhancers.

Fig. 1

Cis-regulatory module recruitment and binding of TFs. (A) Cis-regulatory modules, composed of clustered CREs, regulate transcription by binding TFs and other regulatory proteins. The arrangement of CREs is highly variable and CREs have been identified upstream, downstream and within introns of their target gene. It has also been determined that long distance gene regulation can occur from several megabases away with CREs being brought into proximity through chromatin looping. (B–D) There are three proposed models for how TFs are recruited and assembled at CREs. (B) The flexible billboard model asserts that TFs bind independently to TF-binding motifs found within CRMs resulting in a flexible architecture and grammar for TF binding. (C) The enhanceosome model purports that a TF complex forms prior to being recruited to the CRM. (D) The TF collective model suggests that each CRE can recruit multiple TFs and that while all of these recruited TFs are required, the order of binding and cooperation within the enhanceosome is more flexible. The gene being transcribed is indicated as a rectangle labeled as ‘Gene’, with an arrow representing the direction of transcription. CREs are represented as boxes labeled ‘CREs’, the promoter is represented as a pointed rectangle labeled ‘Pro’, introns are denoted by striped rectangles within the gene target, the terminator is indicated as a black square labeled ‘T’, the RNA polymerase (RNA Pol) is denoted as the shape bound to the promoter and TFs are represented as several shapes labeled ‘TFs’ showing the many different TFs binding together at the CRM. Created in BioRender. Beernink, B. (2024) BioRender.com/j29z706.

Fig. 2

STARR-sequencing (STARR-seq) for plant enhancer identification. This figure illustrates two versions of STARR-seq used for identification of plant enhancers from genomic DNA fragments. Enhancer candidates (Can) were screened in transient reporter assays to determine their enhancer activity. (A) Original STARR-seq (Arnold et al. 2013). Enhancer candidates were cloned into the 3ʹ UTR of constructs containing a minimal cauliflower mosaic virus 35S promoter (MIN 35S), which has weak transcription activity without an enhancer, and GFP, a reporter gene. A no-enhancer negative control (Control) is also included. Constructs were tested in transient reporter assays, such as leaf infiltration or protoplast assays. The messenger RNA is then sequenced, and transcript levels are mapped back to the genome to generate genomic enrichment profiles, identifying candidate enhancer activity. (B) Plant STARR-seq (Jores et al. 2020) is a modified version of the original method, where the candidate enhancers are cloned into the 5ʹ UTR of constructs containing a MIN 35S, GFP and a barcode for sequencing (indicated by the small boxes inside the GFP box). A no-enhancer negative control (Control) is included. Constructs are tested in transient reporter assays. The transcribed barcodes are sequenced and their enrichment relative to the input DNA is calculated giving a distinct enrichment value for each tested enhancer candidate. Enhancers are indicated by boxes labeled ‘Enh’, candidate enhancers are indicated by gray rectangles labeled ‘Can’, the MIN 35S promoter is represented by pointed rectangles with arrows indicating transcription direction and GFP is represented by rectangles labeled ‘GFP’. Created in BioRender. Beernink, B. (2024) BioRender.com/u85z459.

Fig. 3

Enhancer impacts on plant phenotypes despite location variation within plant genomes. (A) The enhancer, Block C, is located upstream of the promoter and the gene being targeted for expression. Block C is demonstrated to upregulate FT gene expression in a reporter assay where an FT–GUS fusion results in significantly more visualized GUS protein after staining. The increased pigment accumulation indicates that higher amounts of the GUS–florigen fusion protein are circulating through the Arabidopsis leaf vasculature in plants with an intact enhancer region, compared to plants where the enhancer was deleted (Adrian et al. 2010). (B) The enhancer, Region C, is located downstream from the gene target, LAS. LAS is a gene involved in Arabidopsis organ boundary determination for leaf and reproductive axials. These findings demonstrate that Region C confers GUS–LAS fusion protein, as visualized by in situ hybridization, significantly higher accumulation of GUS–LAS protein in axial regions when compared to the Region C enhancer deletion mutant (Raatz et al. 2011). (C) Diagram showing the variable locations of Enhancers (Enh) with respect to the gene target. The TE is represented by a triangle, Enhancers are indicated by boxes labeled ‘Enh’, the promoter is represented by a pointed rectangle with the arrow indicating the transcription directionality. The gene target is a box labeled ‘Gene’ with the introns represented as striped boxes, and the transcriptional terminator indicated as a box labeled ‘T’. (D) In some cases, TE insertions provide enhancer elements that will impact plant phenotypes. In the case of apples, it was found that a retrotransposon insertion provided an enhancer element, RedTE, that positively impacted gene expression of the M. domestica (apple; Md) MYB1 gene. In apple trees, where the retrotransposon-inserted enhancer is present, the skin of apples is red due to high levels of pigment accumulation from higher levels of anthocyanin production controlled through MbMYB1 expression. However, in trees where the RedTE enhancer is not present, the skin of the apples remains green (Zhang et al. 2019). (E) Some enhancers are located within introns of target genes. The enhancer, DHS7, upregulated the TRY gene of Arabidopsis impacting trichome formation. When the enhancer is present, trichomes form normally producing three branches, but when the enhancer is deleted, the trichomes develop abnormally, forming four to five branches (Meng et al. 2021). Created in BioRender. Beernink, B. (2024) BioRender.com/c53u001.