The Arabidopsis thaliana rlp mutations revert the ectopic leaf blade formation conferred by activation tagging of the LEP gene

Abstract

Activation tagging of the gene LEAFY PETIOLE (LEP) with a T-DNA construct induces ectopic leaf blade formation in Arabidopsis, which results in a leafy petiole phenotype. In addition, the number of rosette leaves produced prior to the onset of bolting is reduced, and the rate of leaf initiation is retarded by the activation tagged LEP gene. The ectopic leaf blade results from an invasion of the petiole region by the wild-type leaf blade. In order to isolate mutants that are specifically disturbed in the outgrowth of the leaf blade, second site mutagenesis was performed using ethane methanesulphonate (EMS) on a transgenic line that harbours the activation-tagged LEP gene and exhibits the leafy petiole phenotype. A collection of revertant for leafy petiole (rlp lines was isolated that form petiolated rosette leaves in the presence of the activated LEP gene, and could be classified into three groups. The class III rlp lines also display altered leaf development in a wild-type (non-transgenic) background, and are probably mutated in genes that affect shoot or leaf development. The rlp lines of classes I and II, which represent the majority of revertants, do not affect leaf blade outgrowth in a wild-type (non-transgenic) background. This indicates that LEP regulates a subset of the genes involved in the process of leaf blade outgrowth, and that genetic and/or functional redundancy in this process compensates for the loss of RLP function during the formation of the wild-type leaf blade. More detailed genetic and morphological analyses were performed on a selection of the rlp lines. Of these, the dominant rlp lines display complete reversion of (1) the leafy petiole phenotype, (2) the reduction in the number of rosette leaves and (3) the slower leaf initiation rate caused by the activation-tagged LEP gene. Therefore, these lines are potentially mutated in genes for interacting partners of LEP or in downstream regulatory genes. In contrast, the recessive rlp lines exhibit a specific reversion of the leafy petiole phenotype. Thus, these lines are most probably mutated in genes specific for the outgrowth of the leaf blade. Further functional analysis of the rlp mutations will contribute to the dissection of the complex pathways underlying leaf blade outgrowth.