In this work, we investigate morphological differences between Arabidopsis thaliana, which has simple leaves, and its relative Cardamine hirsuta, which has dissected leaves comprising distinct leaflets. With the use of genetics, interspecific gene transfers, and time-lapse imaging, we show that leaflet development requires the REDUCED COMPLEXITY (RCO) homeodomain protein. RCO functions specifically in leaves, where it sculpts developing leaflets by repressing growth at their flanks. RCO evolved in the Brassicaceae family through gene duplication and was lost in A. thaliana, contributing to leaf simplification in this species. Species-specific RCO action with respect to its paralog results from its distinct gene expression pattern in the leaf base. Thus, regulatory evolution coupled with gene duplication and loss generated leaf shape diversity by modifying local growth patterns during organogenesis.

Summary How do genes modify cellular growth to create morphological diversity? We study this problem in two related plants with differently shaped leaves: Arabidopsis thaliana (simple leaf shape) and Cardamine hirsuta (complex shape with leaflets). We use live imaging, modeling, and genetics to deconstruct these organ-level differences into their cell-level constituents: growth amount, direction, and differentiation. We show that leaf shape depends on the interplay of two growth modes: a conserved organ-wide growth mode that reflects differentiation; and a local, directional mode that involves the patterning of growth foci along the leaf edge. Shape diversity results from the distinct effects of two homeobox genes on these growth modes: SHOOTMERISTEMLESS broadens organ-wide growth relative to edge-patterning, enabling leaflet emergence, while REDUCED COMPLEXITY inhibits growth locally around emerging leaflets, accentuating shape differences created by patterning. We demonstrate the predictivity of our findings by reconstructing key features of C. hirsuta leaf morphology in A. thaliana. Video 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...

How the interplay between cell- and tissue-level processes produces correctly proportioned organs is a key problem in biology. In plants, the relative size of leaves compared with their lateral appendages, called stipules, varies tremendously throughout development and evolution, yet relevant mechanisms remain unknown. Here we use genetics, live imaging, and modeling to show that in Arabidopsis leaves, the LATE MERISTEM IDENTITY1 (LMI1) homeodomain protein regulates stipule proportions via an endoreduplication-dependent trade-off that limits tissue size despite increasing cell growth. LM1 acts through directly activating the conserved mitosis blocker WEE1, which is sufficient to bypass the LMI1 requirement for leaf proportionality.

Graphical Abstract Highlights d Identification of genome-wide target genes for the RCO transcription factor d RCO delimits its own expression through autorepression by low-affinity binding d RCO represses local leaf growth via regulating multiple cytokinin (CK)-related genes d RCO negative autorepression fine-tunes CK activity and regulates leaf shape In Brief Hajheidari et al. identify target genes for the RCO homeodomain protein that drove leaf shape diversity. They show that RCO regulates growth via orchestrating homeostasis for the hormone cytokinin and that it also represses its own transcription via low-affinity binding sites. This autorepression helps delimit RCO expression and shape leaf form.

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In the above-mentioned article, we inadvertently omitted two references central to the arguments: Gonzalez et al. (2007) and Chevalier et al. (2011). Both have now been added to the paper, which has been updated online, and can be found on pages 1362 and 1363 in the sentences beginning, "One of the key cell cycle genes showing…" and "However, these findings are in contrast to previous reports…," respectively.

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