A key question in biology is how differences in gene function or regulation produce new morphologies during evolution. Here we investigate the genetic basis for differences in leaf form between two closely related plant species, Arabidopsis thaliana and Cardamine hirsuta. We report that in C. hirsuta, class I KNOTTED1-like homeobox (KNOX) proteins are required in the leaf to delay cellular differentiation and produce a dissected leaf form, in contrast to A. thaliana, in which KNOX exclusion from leaves results in a simple leaf form. These differences in KNOX expression arise through changes in the activity of upstream gene regulatory sequences. The function of ASYMMETRIC LEAVES1/ROUGHSHEATH2/PHANTASTICA (ARP) proteins to repress KNOX expression is conserved between the two species, but in C. hirsuta the ARP-KNOX regulatory module controls new developmental processes in the leaf. Thus, evolutionary tinkering with KNOX regulation, constrained by ARP function, may have produced diverse leaf forms by modulating growth and differentiation patterns in developing leaf primordia.
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We thank S. Hake for 35S::BP and 35S::KN1-GR plasmids and helpful discussions; M. Scanlon for anti-KNOX antibody, J. Long for STM plasmid and advice on in situ hybridization; I. Furner for mutagenesis, T. Rich, S. Hiscock and D. Bailey for help with C. hirsuta identification; Y. Eshed and J. Bowman for cloning vectors, FIL::LhG4 and PHV::LhG4 lines; M. Lenhard for ANT::LhG4; J. Craft for VTOp::BP and VTOP::STM lines; I. Moore for discussions on the LhG4 transcription activation system and S. Langer and M. Anezaki for technical assistance. We also thank J. Langdale for comments on the manuscript, A. Hudson for helpful discussions, J. Baker for photography and Genome and Agricultural Biotechnology for support of library construction. This work was funded by a Biotechnology and Biological Sciences Research Council grant and Oxford University Pump Priming grant to M.T. A.H is the recipient of a University of Oxford Glasstone Research Fellowship and a Balliol College Junior Research Fellowship. We also acknowledge the support of the Gatsby Charitable foundation and the Royal Society.
Sequence comparisons of 5′ upstream regions of STM and BP from Arabidopsis and C. hirsuta. (PDF 187 kb)
Leaflet formation in response to ectopic KNOX activity in Arabidopsis. (PDF 1311 kb)
Predicted C. hirsuta AS1 protein sequence. (PDF 118 kb)
Increased KNOX protein accumulation in C. hirsuta AS1 RNAi leaves. (PDF 103 kb)
Loss of adaxial cell differentiation in chas1-1 leaves. (PDF 334 kb)
Analysis of transcript levels in RNAi lines. (PDF 429 kb)
Primer sequences. (PDF 51 kb)
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