The morphological and physiological basis of delayed pollination overcoming pre‐fertilization cross‐incompatibility in Nicotiana

Delayed pollination is widely used to overcome pre‐fertilization incompatibility, but its regulatory mechanisms are unclear. When Nicotiana tabacum was cross‐pollinated with pollen of N. alata, the incompatibility occurring in the basal 1/4 region of the style (pollinated at anthesis: 0‐day‐old pistil) was overcome by delayed pollination (of 6‐day‐old pistil), and the morphological changes and corresponding physiological basis are explored here. The structure and ultrastructure of the pistil were observed under fluorescence microscopy and transmission electron microscopy. Differentially expressed proteins were screened with a monoclonal antibody chip for Nicotiana, and protein expression and distribution were analysed by immunofluorescence. Cellulase and pectinase activities were tested using enzyme‐linked immunosorbent assay kits. The style of Nicotiana is solid in the basal region and pollen tubes grow in the extracellular spaces (ECM) of the transmitting tissue (TTS) cells. Seven of the 22 identified proteins were cell wall‐associated proteins and were expressed at higher levels during pistil senescence. Cellulase and pectinase activities increased. The TTS cells in the basal 1/4 region of the 0‐day‐old style were polygonal and tightly arranged, with narrow ECM, but these were oval or partially dissolved in the 6‐day‐old pistil, leading to wider ECM and richer secretions. The increased expression of cell wall proteins and enhanced enzyme activity during pistil senescence might partially be responsible for the cells becoming oval and the ECM enlarged, providing the morphological basis for delayed pollination overcoming the pre‐fertilization incompatibility between N. tabacum and N. alata. The transmitting tissue cells becoming oval and the extracellular spaces enlarged in senescent pistils and play a role in overcoming pollen‐pistil incompatibility via delayed pollination.