The DAD Genes: Characterisation of branching controls in Petunia x hybrida

Abstract

Branching is one of the major developmental processes that determine the architecture of a plant. Lateral branching is the outgrowth of axillary buds from the main shoot axis of the plant, and in Petunia hybrida this process is controlled by the DECREASED APICAL DOMINANCE (DAD) genes. The three dad mutants are characterised by increased levels of basal branching, and decreased height when compared to wild-type petunia. Double mutant analyses were carried out to determine whether these genes act in the same pathway to control branching. The dad1dad2 and dad2dad3 double mutants were indistinguishable from the dad2 mutant, which indicated that DAD2 acts in the same pathway as DAD1 and DAD3. Grafting experiments confirmed the action of DAD2 in this pathway, potentially with a role in signal reception or transduction. The dad1dad3 double mutant has an additive phenotype with decreased plant height and delayed flowering compared to the single mutants, indicating that these genes do not act in a simple step-wise fashion. The function of the DAD1 and DAD3 genes was investigated by expression analysis using quantitative RT-PCR (qRT-PCR). The genes were expressed in root and stem and showed upregulation in the stem, but not the roots of the dad mutants, suggesting the involvement of a feedback mechanism in the control of gene expression. The expression of DAD1 and DAD3 in the stem of grafted plants was linked with the branching phenotype not the genotype of the scion. A candidate gene (PhMAX2) was investigated to ascertain whether it was the same locus as DAD2. Results from Southern analyses and qRT-PCR experiments, combined with existing sequencing data, indicated that PhMAX2 is unlikely to encode DAD2. The function of the PhMAX2 gene was investigated by construction RNAi transgenic plants with decreased levels of PhMAX2 transcript, which showed a slight increase in branching and decrease in height compared to wild-type petunia. A model is presented that describes how the DAD genes are thought to act together, as well as with internal and external signals, to control the outgrowth of axillary buds into branches in petunia.