Coupled enhancer and coding sequence evolution of a homeobox gene shaped leaf diversity

Abstract

Here we investigate mechanisms underlying the diversification of biological forms using crucifer leaf shape as an example. We show that evolution of an enhancer element in the homeobox gene REDUCED COMPLEXITY (RCO) altered leaf shape by changing gene expression from the distal leaf blade to its base. A single amino acid substitution evolved together with this regulatory change, which reduced RCO protein stability, preventing pleiotropic effects caused by its altered gene expression. We detected hallmarks of positive selection in these evolved regulatory and coding sequence variants and showed that modulating RCO activity can improve plant physiological performance. Therefore, interplay between enhancer and coding sequence evolution created a potentially adaptive path for morphological evolution.Supplemental material is available for this article.Received September 29, 2016; revised version accepted October 25, 2016. Understanding the genetic basis for evolutionary change is a fundamental problem in biology. Morphological diversity is often underpinned by cis-regulatory divergence of developmental genes and consequent spatiotemporal modification of their expression (Gompel et al. 2005;Hay and Tsiantis 2006;Prud'homme et al. 2006;Carroll 2008;Chan et al. 2010;Frankel et al. 2011;Studer et al. 2011;Arnoult et al. 2013;Rast-Somssich et al. 2015;Indjeian et al. 2016). However, the origin of specific cisregulatory elements underlying morphological diversity is still poorly understood (Rebeiz et al. 2015). For example, it is unclear whether such cis elements tend to arise de novo from rapidly evolving sequences or through the cooption of existing conserved regulatory sequences (Rebeiz et al. 2011;Boyd et al. 2015;Villar et al. 2015). Furthermore, it has not been investigated whether and how coding sequences evolve in concert with regulatory changes to optimize gene function in a new expression domain. Finally, links between regulatory changes underlying morphological change and organismal physiology and fitness remain scarce.Plant leaves present a useful genetic model to tackle these questions because they show substantial morphological variation (Shleizer-Burko et al. 2011;Bar and Ori 2014) and have considerable eco-physiological importance as the major site of photosynthetic carbon fixation in terrestrial ecosystems (Givnish 1978). The REDUCED COMPLEXITY (RCO) gene played a key role in leaf shape diversification in the crucifer family (Sicard et al. 2014;Vlad et al. 2014), to which the reference plant Arabidopsis thaliana belongs. RCO arose through gene duplication and encodes a class I homeobox leucine zipper protein.Its function was discovered in Cardamine hirsuta, where it acts to divide the leaf into distinct leaflets by locally repressing growth at the leaf margin, creating a complex shape. This species-specific activity of RCO arose by neofunctionalization following gene duplication of its ancestral paralog, LMI1, which is conserved in seed plants. Specifically, RCO acquired a novel expression domain within...

Successive duplication-divergence mechanisms at the RCO locus contributed to leaf shape diversity in the Brassicaceae

“…Our expression studies and promoter-swap experiments indicated that both cis-regulatory evolution and intronic or coding sequence polymorphisms underlie the functional divergence of the Capsella RCO paralogs. This is consistent with previous findings in C. hirsuta that demonstrated that ChRCO has diverged from ChLMI1 through both cis-regulatory and protein sequence evolution (Vuolo et al, 2016). The expression of ChRCO, CrRCO-B or NpRCO (which appears to be controlled by the promoter of copy 2 in Neslia owing to the deletion of the NpRCO promoter and NpRCO-B coding sequence as described below) in A. thaliana has similar effects on leaf shape (Vlad et al, 2014) (Figs 2 and 6), suggesting that the At this stage, it is still difficult to firmly conclude whether the functional divergence of the three RCO paralogs reflects the partitioning of the ancestral function or the neo-functionalization of the incipient copies.…”

“…The functional diversification of RCO-A and RCO-B was accompanied by the acquisition of very specific expression patterns, suggesting that cis-regulatory evolution underlies their diversification. The functional specialization of C. hirsuta RCO (in position 2 in the RCO cluster) from ChLMI1 was previously reported to be the result of both cis-regulatory element and protein coding sequence evolution (Vlad et al, 2014;Vuolo et al, 2016). We confirmed that the promoter of the Capsella CrRCO-B gene also carries all the specific information required to induce the formation of lyrate lobed leaves ( Fig.…”

Enhancer evolution and the origins of morphological novelty

“…Diversification of RCO from LMI1 arose through cis-regulatory evolution, which generated a novel and specific RCO expression domain at the base of developing leaflets in a region pivotal for shape determination. To investigate how this occurred, Vuolo et al [38] discovered that a leaf-margin enhancer of LMI1 which drives gene expression distally in leaf primordia was repurposed in the RCO paralog to drive expression proximally, flanking the emerging leaflets (Figure 3B). They also showed that a single amino acid substitution reduced RCO protein stability, which suppressed the potential pleiotropic effects of its altered expression.…”

Conservation vs divergence in LEAFY and APETALA1 functions between Arabidopsis thaliana and Cardamine hirsuta

“…Both species belong to the Brassicaceae family, diverged c. 32 Ma and are reproductively isolated Gan et al, 2016). Comparative genetic analyses in these species have successfully identified molecular changes that underlie phenotypic differences that are of evolutionary significance, such as leaf shape and seed dispersal (Hay & Tsiantis, 2006;Barkoulas et al, 2008;Vlad et al, 2014;Hofhuis et al, 2016;Vuolo et al, 2016).…”