Impact of segmental chromosomal duplications on leaf size in the grandifolia-D mutants of Arabidopsis thaliana

The number of cells in an organ is a major factor that specifies its size. However, the genetic basis of cell number determination is not well understood. To obtain insight into this genetic basis, three grandifolia-D (gra-D) mutants of Arabidopsis thaliana were characterized that developed huge leaves with two to three times more cells than the wild-type. Genetic and microarray analyses showed that a large segmental duplication had occurred in all the gra-D mutants, consisting of the lower part of chromosome 4. In the duplications, genes were found that encode AINTEGUMENTA (ANT), a factor that extends the duration of cell proliferation, and CYCD3;1, a G(1)/S cyclin. The expression levels of both genes increased and the duration of cell proliferation in the leaf primordia was extended in the gra-D mutants. Data obtained by RNAi-mediated knockdown of ANT expression suggested that ANT contributed to the huge-leaf phenotype, but that it was not the sole factor. Introduction of an extra genomic copy of CYCD3;1 into the wild-type partially mimicked the gra-D phenotype. Furthermore, combined elevated expression of ANT and CYCD3;1 enhanced cell proliferation in a cumulative fashion. These results indicate that the duration of cell proliferation in leaves is determined in part by the interaction of ANT and CYCD3;1, and also demonstrate the potential usefulness of duplication mutants in the elucidation of genetic relationships that are difficult to uncover by standard single-gene mutations or gain-of-function analysis. We also discuss the potential effect of chromosomal duplication on evolution of organ size.