BONOBO transcription factors positively regulate STICKY GENERATIVE CELL for generative cell internalisation in Arabidopsis.

key messageGenerative cell internalisation establishes distinctive 'cell-within-a-cell' pollen organization in angiosperms. Comparative and transgenic expression analyses provide valuable insights into the cytological and genetic regulation underlying generative cell internalisation in Arabidopsis. For double fertilisation in flowering plants, a three-celled male gametophyte (pollen grain) delivers two non-motile sperm cells to the female gametes through a rapidly elongating pollen tube. This process is facilitated by a distinctive'cell-within-a-cell' pollen organisation, where the smaller generative cell becomes internalised into the larger vegetative cytoplasm through precisely regulated morphogenetic process and callose dynamics. However, the genetic mechanisms underlying generative cell internalisation remain unclear. Therefore, this study aims to comparatively analyse Arabidopsis wild-type with two different mutants that exhibit immobilised generative cells from the loss-of-function of DUF707 STICKY GENERATIVE CELL (SGC) and bHLH transcription factors BONOBO (BNB1) and BNB2, respectively. The results show that wild-type generative cells internalise through four successive steps. In contrast, bnb1;bnb2 mutants fail to progress to step 3 due to incomplete dissolution of the hemispherical callose and sgc-1 mutant proceeds to step 3 but arrests before step 4 with the intine-side callose dissolution disturbed. Moreover, transgenic analyses employing SGC promoter reporters and various BNB expression constructs revealed that SGC expression is positively regulated by BNB transcription factors. These findings enhance the understanding of the cytological and genetic regulation involving BNB transcription factors and downstream components, including SGC, during generative cell internalisation.