Most varieties of persimmon (Diospyros kaki Thunb.) are gynoecious, while just a few are either monoecious, androgynomonoecious, or androecious. Persimmon flowers initially contain the original androecium and gynoecium followed by arrest of either pistil or stamen primordia before maturity. Abortion of inappropriate primordium in persimmon may be related to programmed cell death (PCD). To test this hypothesis, hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, cyt-c immunohistochemistry (IHC) assay, transmission electron microscopy (TEM) observation, and real-time quantitative polymerase chain reaction (RT-qPCR) were used to clarify the occurrence and molecular regulatory mechanism of PCD in male and female floral buds during the 14 days prior to the second crucial morphological stage when inappropriate sexual primordia were arrested to form unisexual flowers. Accordingly, dead cells in inappropriate sex organs were largely accumulated during the microsporocyte and macrosporocyte period of male and female floral buds, respectively. This may explain the abortion of inappropriate sex organs, leading to unisexual flowers. PCD is necessary for normal growth and development in persimmons, as dead cells could also be observed in the normal flower organs. High levels of a gene homologous to AMC9 may have accelerated the arrest of the pistil primordium during differentiation, leading to male unisexual flowers, and high levels of genes homologous to MeGI, BAG5, AifA, and HSP70 in female floral buds were positively correlated with the arrest of stamen primordium. Future studies may try to transform unisexual flowers into hermaphroditic flowers by the regulation of PCD artificially, which will be helpful to the controlled pollination experiments.

It is reported that the production of floral sexual phenotype in hexaploid monoecious persimmon (Diospyros kaki) is closely related to a pseudogene called OGI, and a short interspersed nuclear element (SINE)-like insertion (named Kali) in the OGI promoter leads to the gene silence. As a result, DNA methylation level of MeGI promoter determines the development of male or female flowers. However, the molecular mechanism in androecious D. kaki, which only bear male flowers, remains elusive. Here, real-time quantitative polymerase chain reaction (RT-qPCR), molecular cloning, and bisulfite PCR sequencing technique were carried out using 87 materials, including 56 androecious resources, 15 monoecious, and 16 gynoecious cultivars, to investigate the performance of OGI and MeGI on the specific androecious type of D. kaki in China. In conclusion, the Kali insertion was exactly located in the OGI promoter region, and the OGI gene and the Kali sequence were existing and conserved in androecious D. kaki. Meanwhile, we also demonstrated that the MeGI gene was widespread in our investigated samples. Ultimately, our result convincingly provided evidence that the low expression of OGI is probably ascribed to the presence of Kali displaying strong methylation in the OGI promoter, and low expression of MeGI, as well as high DNA methylation level, in the promoter was closely connected with the production of male flowers; this result was consistent with the monoecious persimmon model. Our findings provide predominant genetic aspects for investigation into androecious D. kaki, and future perfecting the sex-determining mechanisms in persimmon.