Abstract
Several hundred genes are required for embryonic and gametophytic development in the model plant Arabidopsis thaliana, as inferred from the lethality of their mutations. Despite many of these genes are expressed throughout the plant life cycle, the corresponding mutants arrest at early stages, preventing the study of their post-embryonic functions by conventional methods. Clonal analysis represents an effective solution to this problem by uncovering the effects of embryo-lethal mutations in sectors of mutant cells within an otherwise normal adult plant. In this pilot study, we have evaluated the suitability of two sector induction methods for the large-scale study of the post-embryonic effects of embryo-lethal (emb) mutations in Arabidopsis. In line with the interests of our laboratory, we selected 24 emb mutations that damage genes that are expressed in wild-type vegetative leaves but whose effects on leaf development remain unknown. For the induction of mutant sectors in adult plants, we followed one approach based on the X-ray irradiation of ‘cell autonomy’ (CAUT) lines, and another based on the site-specific excision of transgenes mediated by Cre recombinase. We conclude that both methods are time-consuming and difficult to scale up, being better suited for the study of emb mutations on a case-by-case basis.
Highlights
Mutational approaches have greatly advanced our understanding of developmental processes in plants and animals
In an attempt to select an efficient strategy that is suitable for the systematic identification of essential genes that function post-embryonically, we have carried out pilot experiments using two different approaches aimed at inducing somatic sectors that express the mutant phenotype, one based on the use of cell autonomy’ (CAUT) lines[14], and another based on the use of the Cre-loxP site-specific recombination system[7]
We focused on a subset of 24 EMBRYO DEFECTIVE (EMB) genes selected from the SeedGenes database, which includes comprehensive information on the embryonic lethal genes of Arabidopsis[15]
Summary
Mutational approaches have greatly advanced our understanding of developmental processes in plants and animals. By focusing on viable mutations, these screenings are likely to have missed many genes that play important post-embryonic roles, because they are essential in early developmental stages and there are not viable alleles to study. Some examples are the angulata[1] (anu1-1), anu[], anu[] and scabra[1] (sca1-1) mutants of Arabidopsis thaliana (hereafter, Arabidopsis), identified in a large-scale screen for viable mutants with abnormal leaf shape, size and pigmentation, which were later found to be hypomorphic alleles of the SECA2, EMBRYO DEFECTIVE 2737 (EMB2737), NON-INTRINSIC ABC PROTEIN 14 (NAP14) and EMB3113 genes[1,2,3]. By inducing mutant sectors in phenotypically wild-type plants, clonal analysis has helped researchers to answer questions regarding the phenotypic effects caused by the complete inactivation of embryo-lethal (EMB) genes in the tissues of an adult plant, the site of action of gene products, the cell autonomy and the cell lethality of lethal mutations. We tested two different strategies, one involving the use of X-rays and CAUT (cell autonomy) lines, and another based on the site-specific excision of transgenes mediated by Cre recombinase
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