Leaf phenomics: a systematic reverse genetic screen for Arabidopsis leaf mutants

Abstract

SUMMARYThe study and eventual manipulation of leaf development in plants requires a thorough understanding of the genetic basis of leaf organogenesis. Forward genetic screens have identified hundreds of Arabidopsis mutants with altered leaf development, but the genome has not yet been saturated. To identify genes required for leaf development we are screening the Arabidopsis Salk Unimutant collection. We have identified 608 lines that exhibit a leaf phenotype with full penetrance and almost constant expressivity and 98 additional lines with segregating mutant phenotypes. To allow indexing and integration with other mutants, the mutant phenotypes were described using a custom leaf phenotype ontology. We found that the indexed mutation is present in the annotated locus for 78% of the 553 mutants genotyped, and that in half of these the annotated T-DNA is responsible for the phenotype. To quickly map non-annotated T-DNA insertions, we developed a reliable, cost-effective and easy method based on whole-genome sequencing. To enable comprehensive access to our data, we implemented a public web application named PhenoLeaf (http://genetics.umh.es/phenoleaf) that allows researchers to query the results of our screen, including text and visual phenotype information. We demonstrated how this new resource can facilitate gene function discovery by identifying and characterizing At1g77600, which we found to be required for proximal-distal cell cycle-driven leaf growth, and At3g62870, which encodes a ribosomal protein needed for cell proliferation and chloroplast function. This collection provides a valuable tool for the study of leaf development, characterization of biomass feedstocks and examination of other traits in this fundamental photosynthetic organ.

Distributed phenomics with the unPAK project reveals the effects of mutations

“…Phenotype datasets using deletion or insertion mutant collections in model organisms are a well-established and rich resource for characterizing genes of unknown function (Winzeler et al, 1999). These datasets have been used to effectively test hypotheses across disciplines, including evolutionary biology, cell and molecular biology, systems biology, network biology and genomics (Winzeler et al, 1999;Hirsh and Fraser, 2001;Giaever et al, 2002;Gu et al, 2003;Chern et al, 2007;Bell, 2010;O'Malley and Ecker, 2010;Ramani et al, 2012;Lehner, 2013;Wilson-S anchez et al, 2014). Despite these efforts, surveys of mutant collections have often detected few phenotypic consequences of gene disruption.…”

“…A study of 4000 insertion lines estimated that < 4% of lines resulted in discernibly altered phenotypes in a survey of leaf shape and related traits (Kuromori et al, 2006;Hanada et al, 2009). More recently, a screen of 706 mutants had a similar rate of discovery when evaluating categorical leaf mutant traits (3.6%; Wilson-S anchez et al, 2014). The general lack of detectable phenotypes differing from wild-type in many screens of knockout mutations has been termed the 'knockout paradox' (Papp et al, 2011;Albalat and Cañestro, 2016).…”

A Journey Through a Leaf: Phenomics Analysis of Leaf Growth inArabidopsis thaliana

“…Next to forward genetic screens, a large-scale reverse genetics study on a collection of gene-indexed insertional mutants of Arabidopsis has recently been performed to identify genes involved in leaf development (Wilson-Sánchez et al, 2014). This screen resulted in the identification of 706 mutants exhibiting a leaf phenotype, such as changes in rosette and/or leaf lamina size, leaf shape and color.…”

Members of the DEAL subfamily of the DUF1218 gene family are required for bilateral symmetry but not for dorsoventrality in Arabidopsis leaves

“…We previously screened the Salk collection of homozygous T-DNA lines (Alonso et al, 2003) for mutants with altered leaf form (Wilson-S anchez et al, 2014). Among the 706 viable leaf mutants we identified, only one had leaves that clearly deviated from bilateral symmetry: the SALK_047972 line ( Fig.…”