Evolution of calyx diversity in angiosperms: a focus on transcriptomic repatterning mechanisms underlying inflated fruiting calyx within Solanaceae

The angiosperm calyces display considerable diversity and have adaptive functions. However, the evolutionary trajectories and underlying mechanisms of calyx morphological diversity remain unclear. In this study, ancestral state reconstruction revealed that the abscised calyx was ancestral; however, most extant angiosperms exhibited persistent calyces showing notable variation in size. Remarkably, the Solanaceae family may represent a miniature reflecting the calyx diversity of angiosperms. Distinct from Solanum and Capsicum, Physalis fruits featured a morphological novelty known as inflated calyx syndrome (ICS). To reveal the molecular repatterning events underlying ICS formation, we conducted time-course transcriptomic comparisons on developing calyces of ICS species (Physalis floridana) and non-ICS species (Capsicum annuum and two Solanum species), and detected that variations in heterometric expression and alternative splicing were predominant across these species. Moreover, two Physalis-calyx highly expressed genes respectively encoding PHYSALIS ORGAN SIZE 4 (POS4) and POS5 were knocked down and out using virus-induced gene silencing and CRISPR/Cas9 technologies, and the resulting genetically modified P. floridana plant lines displayed a significant reduction in ICS size. Furthermore, when compared with Solanum and Capsicum, heterotopically expressed genes in Physalis calyx relative to berry were mainly enriched for functions in photosynthesis and responses to stimuli, thereby supporting the hypothesis that the inflated fruiting calyx may have partitioned and exapted functions originally associated with berry. This work elucidates the calyx evolutionary pattern of angiosperms as well as transcriptomic repatterning mechanisms that may govern both developmental and functional evolution of fruiting calyx inflation within Solanaceae, thereby providing insights into plant morphological evolution.