The RAF-like mitogen-activated protein kinase kinase kinases RAF22 and RAF28 are required for the regulation of embryogenesis in Arabidopsis.

In Arabidopsis, embryonic development follows a stereotypical pattern of cell division. Although many factors have been reported to participate in establishment of the proper embryonic pattern, the molecular mechanisms underlying pattern formation are unclear. In this study we showed that RAF22 and RAF28, two RAF-like mitogen-activated protein kinase kinase kinases (MAPKKKs) in Arabidopsis, are involved in the regulation of embryogenesis. The double knockout mutant of RAF22 and RAF28 was embryo lethal. A large proportion of the raf22-/- raf28+/- mutant embryos exhibited various defects, including disordered proembryo cell divisions, disruption of the bilaterally symmetrical structure, abnormally formative divisions of hypophysis and exaggerated suspensor growth. Whereas the kinase active form of RAF22 could complement these embryonic aberrant phenotypes, the kinase inactive form could not. The restrictive expression of the basal cell fate marker WOX8 in the abnormally dividing suspensor cells and the apical cell linage marker WOX2 in the abnormal proembryos indicated that apical and basal cell fates were unchanged in the abnormal embryos. The polar distribution of the auxin maxima and the PIN1 and PIN7 auxin transporters was markedly altered in the abnormal embryos. Our results suggest that RAF22 and RAF28 are important components of embryogenesis and that auxin polar transport may be involved in this regulation.