Premise of research. According to the neosynangial hypothesis, sterilization of megasporangia and compaction of fertile appendages bearing megasporangia were critical steps for integument evolution. Ovules of bell1 (bel1) mutants in Arabidopsis thaliana can form ectopic nucelli in the chalazal domain instead of inner and outer integuments, while distances between ectopic nucelli expand when class III homeodomain-leucine zipper (HD-ZIPIII) genes are compromised in addition to bel1. These phenotypes imply that BEL1 and HD-ZIPIII genes would be participants in the sterilization and compaction steps, respectively. However, this interpretation is challenged by the observation that bel1 ovules also form an outgrowth in the chalazal domain. The outgrowth finally develops into a carpel-like structure (i.e., the ectopic nucelli would be formed on the placenta of this ectopic carpel). We revisited bel1 phenotypes to rule out this possibility and inferred how these genes contributed to integument evolution.

Methodology. We observed development and expression of a carpel identity marker CRABS CLAW (CRC) in bel1 ovules. We compared expression of SPOROCYTELESS/NOZZLE (SPL/NZZ), a master regulator of nucellus formation, between bel1 and wild-type carpels by quantitative real-time polymerase chain reaction. We observed accumulation of PIN-FORMED 1 (PIN1) protein in triple mutants of HD-ZIPIII genes.

Pivotal results. Morphological observation, as well as expression of CRC, suggested that ectopic nucelli are formed before induction of carpel identity. BEL1 represses SPL/NZZ expression. PIN1 expression was not altered in triple mutants of HD-ZIPIII genes, suggesting that they do not repress the nucellus-promoting pathway(s).

Conclusions. The bel1 phenotype suggests that seed plant ovules have a repressed ability to form multiple nucelli, similar to fertile appendages of progymnosperms. BEL1 would have promoted sterilization of megasporangia by repressing SPL/NZZ. HD-ZIPIII genes would have controlled spacing between megasporangia in fertile appendages by regulating the size of the meristematic area rather than by repressing formation of extra megasporangia.