Role of basic helix-loop-helix transcription factors in formation of pegged rhizoids in the liverwort Marchantia polymorpha

Abstract

When land plants diverged from their aquatic ancestors, various adaptations were necessary for survival in the more arid terrestrial environment. These include the development of a waterproof cuticle, presence of gas exchange pores such as stomata, production of a multicellular embryo able to produce numerous spores from a single fertilisation event, and efficient water transport systems. In Arabidopsis thaliana, the seed-specific gene ZHOUPI (ZOU) controls both cuticle formation in the embryo, and also cell separation and death of the surrounding endosperm tissue to allow the growth of the embryo. ZOU encodes a basic helix-loop-helix (bHLH) transcription factor and it acts as a heterodimer with another bHLH protein, INDUCER OF CBF EXPRESSION1 (ICE1). Consequently, ZOU and ICE1 share common mutant phenotypes in Arabidopsis seed. However, ICE1 is expressed more broadly than ZOU, and unlike ZOU it also regulates stomatal fate in leaves. The ZOU and ICE1 genes are conserved all land plants, including in early diverging lineages that lack seed, endosperm and stomata. The aim of my thesis was to use genetic and molecular analysis to determine the function of ZOU and ICE genes in the liverwort Marchantia polymorpha. The Marchantia genome encodes two homologues of Arabidopsis ZOU, MpZOU1 and MpZOU2. In addition, there are two ICE1 genes, MpICE1a and MpICE1b. Using yeast two hybrid assays, I found that the interaction of ZOU and ICE1 proteins in conserved in Marchantia. I also found that both MpZOU1 and MpZOU2 could complement the Arabidopsis zou mutant phenotype when expressed in transgenic seed. This suggested that the molecular activity of the ZOU/ICE1 heterodimer is conserved. To determine the function of the Marchantia ZOU orthologues, I characterised their expression patterns, inactivated them using CRISPR/Cas9 genome editing and also mis-expressed them. The expression patterns of MpZOU1 and MpZOU2 are distinct, with MpZOU1 strongly expressed in the ventral midrib, whereas MpZOU2 is mainly expressed dorsally in epidermis and air chambers. Mutant analysis showed that MpZOU1 is needed for formation of pegged rhizoids, which are distinguished from smooth rhizoids by the presence of secondary cell wall thickenings (pegs) and the fact that they undergo programmed cell death. Consistent with pegged rhizoids being important for water transport, reproductive structures shrivelled and dried out in mutants. In addition, mis-expression experiments suggested that MpZOU1 directs smooth rhizoids to differentiate as pegged rhizoids. Preliminary results suggested that mpzou2 mutants had normal pegged rhizoids but showed defects in cuticle formation in gemmae. My results suggest that the ZOU/ICE1 partnership was present in the earliest land plants and is vital for several adaptations to life on land. In order to determine the target genes involved in pegged rhizoid formation, transcriptomic experiments were used to compare mutants and wild type. The results showed that genes involved in cell wall modification and apoptosis are down regulated in mutants lacking pegged rhizoids. Further analysis of the target genes may help address whether the role of ZOU genes in pegged rhizoid formation is novel or has common features with cuticle formation or endosperm breakdown in angiosperms.