A suppressor screening of jaw-D, a microRNA overexpressing mutant

MicroRNAs (miRNAs) are small RNAs with regulatory function in both plants and animals. Their mode of action requires interaction with target mRNAs through complementary basepairing. In plants, most miRNAs seem to guide their mRNA targets to cleavage, causing downregulation of target transcripts. miR-JAW (miR-319a), like several other miRNAs, plays important roles in plant development. The jaw-D mutant, which constitutively overexpresses miR-JAW, shows pleiotropic phenotypes, including epinastic cotyledons, crinkly leaves and a delay in flowering time. This is due to the simultaneous downregulation of five genes belonging to the TCP class of plant-specific transcription factors, which are thought to be involved in the regulation of cell proliferation. To gain further insight into the molecular mechanisms responsible for miR-JAW activity and function, we carried out a suppressor screen in jaw-D background. We mutagenized 20.000 jaw-D seeds with EMS and screened the M2-population for plants in which the jaw-D associated phenotypes are suppressed. 50 putative suppressors were isolated. In some, all phenotypes of jaw-D mutants are affected, while in others only a subset is affected, like leaf shape or flowering time. RT-qPCR of the TCPs allows a further classification of the suppressors, e.g. a low TCP level would imply that the suppressor acts downstream of the transcription factors. We will describe the phenotypic and molecular characterization of several suppressor mutants