Quantitative methods in like-for-like comparative analyses of Aphanorrhegma (Physcomitrella) patens phyllid development

Abstract

Introduction. Aphanorrhegma (Physcomitrella) patens (Hedw.) Lindb. is an attractive model system for comparative analyses of leaf development because it evolved leaves (phyllids) independently to flowering plants, yet its genome contains homologues of many gene families that regulate angiosperm leaf development. In addition, A. patens phyllids are primarily a single cell layer thick, making it simple to identify the cellular basis of defects that perturb shape. Identification of gene functions in shape determination depends on like-forlike comparison of mutant versus wild-type plants. Key results. Here we show that, if heteroblasty is not perturbed, such comparisons should use phyllid L13 or above in the heteroblastic series, and fully expanded phyllids above P7 in the developmental series. Using a quantitative approach, we show that heteroblastic size variation reflects differences in cell proliferation rather than cell size and shape. A comparison of control to pinA pinB mutant phyllid development verifies that PIN proteins promote cell proliferation and suppress expansion to determine phyllid shape. Conclusion. The results and approach that we have generated will be applicable to any study of A. patens phyllid development to reveal the cellular basis of phyllid size and shape variations.