The heterotrimeric protein phosphatase 2A (PP2A) complex comprises a catalytic subunit and regulatory A and B subunits that modulate enzyme activity and mediate interactions with other proteins. We report here the results of a systematic analysis of the Arabidopsis (Arabidopsis thaliana) regulatory A subunit gene family, which includes the ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1 (RCN1), PP2AA2, and PP2AA3 genes. All three A subunit isoforms accumulate in the organs of seedlings and adult plants, suggesting extensive overlap in expression domains. We have isolated pp2aa2 and pp2aa3 mutants and found that their phenotypes are largely normal and do not resemble that of rcn1. Whereas rcn1 pp2aa2 and rcn1 pp2aa3 double mutants exhibit striking abnormalities in all stages of development, the pp2aa2 pp2aa3 double mutant shows only modest defects. Together, these data suggest that RCN1 performs a cardinal role in regulation of phosphatase activity and that PP2AA2 and PP2AA3 functions are unmasked only when RCN1 is absent.

In Arabidopsis thaliana, gene expression level polymorphisms (ELPs) between natural accessions that exhibit simple, single locus inheritance are promising quantitative trait locus (QTL) candidates to explain phenotypic variability. It is assumed that such ELPs overwhelmingly represent regulatory element polymorphisms. However, comprehensive genome-wide analyses linking expression level, regulatory sequence and gene structure variation are missing, preventing definite verification of this assumption. Here, we analyzed ELPs observed between the Eil-0 and Lc-0 accessions. Compared with non-variable controls, 5 0 regulatory sequence variation in the corresponding genes is indeed increased. However, B42% of all the ELP genes also carry major transcription unit deletions in one parent as revealed by genome tiling arrays, representing a 44-fold enrichment over controls. Within the subset of ELPs with simple inheritance, this proportion is even higher and deletions are generally more severe. Similar results were obtained from analyses of the Bay-0 and Sha accessions, using alternative technical approaches. Collectively, our results suggest that drastic structural changes are a major cause for ELPs with simple inheritance, corroborating experimentally observed indel preponderance in cloned Arabidopsis QTL.

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.