Pollen grains show an enormous variety of aperture systems. What genes are involved in the aperture formation pathway and how conserved this pathway is in angiosperms remains largely unknown. INAPERTURATE POLLEN1 (INP1) encodes a protein of unknown function, essential for aperture formation in Arabidopsis, rice and maize. Yet, because INP1 sequences are quite divergent, it is unclear if their function is conserved across angiosperms. Here, we conducted a functional study of the INP1 ortholog from the basal eudicot Eschscholzia californica (EcINP1) using expression analyses, virus-induced gene silencing, pollen germination assay, and transcriptomics. We found that EcINP1 expression peaks at the tetrad stage of pollen development, consistent with its role in aperture formation, which occurs at that stage, and showed, via gene silencing, that the role of INP1 as an important aperture factor extends to basal eudicots. Using germination assays, we demonstrated that, in Eschscholzia, apertures are dispensable for pollen germination. Our comparative transcriptome analysis of wild-type and silenced plants identified over 900 differentially expressed genes, many of them potential candidates for the aperture pathway. Our study substantiates the importance of INP1 homologs for aperture formation across angiosperms and opens up new avenues for functional studies of other aperture candidate genes.

In the last decade, certain genes involved in pollen aperture formation have been discovered. However, those involved in pollen aperture shape remain largely unknown. In Arabidopsis, the interaction during the tetrad development stage of one member of the ELMOD protein family, ELMOD_E, with two others, MCR/ELMOD_B and ELMOD_A, can change the morphology of apertures from colpus (elongated) to pore (round). Here, comparative transcriptome analysis is used to identify candidate genes involved in the determination of pollen aperture morphology in Papaveraceae (order Ranunculales). Furthermore, the role of ELMOD genes in the genetic determinism of aperture shape was tested by comparative analysis of their expression levels using RNA-seq data and RT-qPCR. Two pairs of species belonging to two different subfamilies were used. Within each pair, one species has colpate pollen and the other porate (Fumarioideae—Dactylicapnos torulosa, 6-colpate, and Fumaria bracteosa, pantoporate; Papaveroideae—Eschsholzia californica, 5–7 colpate, and Roemeria refracta, 6-porate). The transcriptomes were obtained at the tetrad stage of pollen development. A total of 531 DEGs were found between the colpate and porate pollen species groups. The results from RNA-seq and RT-qPCR indicate that pollen aperture shape is not determined by the relative expression levels of ELMOD family genes in Papaveraceae. However, genes related to callose wall formation or cytoskeleton organisation were found, these processes being involved in pollen aperture formation. In addition, transcriptomes from anthers with pollen during the tetrad stage of three species (D. torulosa, R. refracta, and F. bracteosa) were obtained for the first time. These data will be available for further studies in the field of floral evolution and development.