Genetic dissection reveals distinct roles for the transcription factor ZHOUPI in controlling arabidopsis endosperm cell death and embryonic cuticle development

Abstract

Angiosperm seed development requires co-ordinated development of the embryo and a second zygotic tissue, the endosperm. In Arabidopsis thaliana, the endosperm is ephemeral and is largely consumed by the embryo during seed development. In addition to a role in embryo nutrition, it is also likely that the endosperm may play a more direct role in signalling to the embryo to regulate development. Despite their importance for embryo development, these processes are very poorly understood. The ZHOUPI (ZOU) gene provides an important tool to address these problems. Firstly, ZOU likely regulates endosperm breakdown. Whereas wild-type seed have a single layer of endosperm at maturity, zou seed has a large persistent endosperm and a correspondingly small embryo. The small zou embryo does not fill the seed so that the seed shrivels as it desiccates during maturation. Secondly, zou embryos have defects in their cuticle, so that the endosperm adheres to the embryo throughout seed development. After seed germination, zou cotyledons develop holes in their epidermis as they expand, probably due to the defects in the cuticle. ZHOUPI (ZOU) encodes a bHLH transcription factor and is expressed in the embryo surrounding region (ESR) of endosperm but not in the embryo itself. The role of ZOU in cuticle development is partly mediated by the ABNORMAL LEAF SHAPE1 (ALE1) gene. Thus, ale1 mutants also show defects in embryonic cuticle development and ALE1 is specifically expressed in ESR in a ZOU-dependent fashion. It was unclear whether the effects of ZOU upon embryo development are an indirect consequence of the persistent endosperm mechanically impeding embryo expansion, or rather reflect a more direct role of the ESR in signalling to the embryo. The main aims of this thesis were 1) to provide evidence that ZOU regulates endosperm cell death and 2) to test whether ZOU function in controlling endosperm cell death could be separated from that in embryonic epidermal cuticle development. To achieve this goal, 1) TUNEL assays were performed in the seeds to confirm the zou endosperm cell death phenotype, 2) ALE1 expression in the ESR in zou mutants was rescued using the ZOU-independent AtSUC5 promoter to investigate whether one or both of zou phenotypes were complemented, 3) Candidate ZOU target genes were validated and characterized to determine their functions in endosperm cell death and/or embryonic epidermal cuticle development. The TUNEL assays revealed that zou mutants display less DNA fragmentation in the ESR than that of the wild-type, but that zou did not have defects in cell death outside the seeds suggesting ZOU specifically regulated endosperm cell death. The AtSUC5::ALE1 transgene partially rescued zou defects in epidermal cuticle but not in endosperm cell death. This shows that the defects in the zou cuticle are not caused by the defective endosperm, rather zou has distinct, separable functions. Lastly, I characterised several novel ZOU targets and showed that RGP3 may be a direct ZOU target as it is expressed in ESR in ZOU dependent fashion, whereas RGP4 is likely indirect as it is expressed in the testa and up-regulated in zou mutants. In conclusion, ZOU has independent roles in endosperm cell death and embryonic epidermal cuticle development. Because ALE1, which largely mediates the role in cuticle development, is less widely conserved than is ZOU, the role in promoting endosperm cell death may be the ancestral function of ZOU.