CAUT lines: a novel resource for studies of cell autonomy in Arabidopsis

Abstract

SummaryPlant development is critically dependent on the interactions between clonally unrelated cell layers. The crosstalk between layers can be addressed by studies of cell autonomy. Cell autonomy is a property of genetic mosaics composed of cells of differing genotypes. Broadly, if the phenotype of a mutant tissue reflects only its genotype and is unaffected by the presence of wild-type tissue, the trait is cell-autonomous. Conversely, if the phenotype of a mutant tissue reflects that of wild-type tissue in the mosaic, the trait is non-autonomous. Here we report a novel, versatile and robust method for studies of cell autonomy in Arabidopsis. Cell autonomy (CAUT) lines consist of a collection of homozygous stocks, each containing one of 76 mapped T-DNA inserts, each of which corrects the yellow ch-42 mutant to green (CH-42) by complementation. This has the effect of translocating the colour marker to 76 new locations around the genome. X-irradiation of heterozygous CAUT line seeds results in yellow sectors, with loss of the CH-42 transgene and adjacent wild-type genes. This property can be used to remove the wild-type copy of developmental genes in appropriate heterozygotes, resulting in yellow (ch-42) sectors that are hemizygous for the trait of interest. Such sectors can provide insight into cell autonomy. Experiments using the ap1, ap3, ag and clv1 mutants show that CAUT lines are useful in the study of cell autonomy.

Suitability of two distinct approaches for the high-throughput study of the post-embryonic effects of embryo-lethal mutations in Arabidopsis

“…In an attempt to select an efficient strategy that is suitable for the systematic identification of essential genes that also 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 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 ( http://www.seedgenes.org/ ), which includes comprehensive information on the embryonic lethal genes of Arabidopsis 15 .…”

“…For the induction of marked somatic sectors in Arabidopsis, we initially took advantage of the availability of CAUT lines with insertions located on every chromosome arm 14 . Thirteen different EMB genes (Table 1 ) were selected based on the availability of suitable CAUT lines carrying an insertion of the CHLORATA-42 ( CH-42 ) gene located between the EMB gene and the centromere of the corresponding chromosome.…”

“…Any developmental or other visible phenotypes occurring specifically in the yellow sectors can be attributed to the post-embryonic effects of the corresponding emb mutation only if they are not observed in the irradiated control families. Because the cells in the L1 layer are colorless and those in the L3 contribute comparatively little to most organs, the ch-42 yellow phenotype is best scored in the cells of the L2 layer, making this marker most useful for the study of genes that function in this layer 14 .…”

“…On the one hand, water-imbibed seeds were subjected to a dosage of 1000 rad (10 Gy) based on previous reports from the Arabidopsis and maize literature 6 . On the other, dry seeds received a dosage of 16000 rad (160 Gy), as previously described 14 . The irradiation of dry seeds allowed us to stagger the sowing of the irradiated families.…”

“…3d–f ), a frequency that is roughly similar to the frequency reported by Furner et al . 14 . In the six families that exhibited a clear distortion of the Mendelian segregation of the yellow phenotype caused by ch-42 , we found somatic sectors in both types of irradiated families (segregating and not segregating the emb mutation; Fig.…”