Epsilon Open Archive

Abstract

The early plant embryo is divided into two domains with contrasting fates: apical embryo proper (in angiosperms) or embryonal mass (in gymnosperms) and basal suspensor. While apical domain is composed of proliferating cells and develops to plant, the suspensor consists of terminally-differentiated cells and functions as a conduit of growth factors to the growing apical domain followed by elimination through programmed cell death (PCD). The strict balance between cell division and PCD in the two domains is critical for correct embryo patterning and alteration of this balance can lead to lethality. The aim of this thesis was to identify and functionally characterise new molecular components participating in the regulation of cell division and death in plant embryo.

Cohesin is a multi-protein complex with important role in DNA replication and
sister chromatid cohesion and separation. In non-plant species, cohesin is loaded on chromatin by the Scc2-Scc4 complex. Arabidopsis thaliana homologue of Scc4 (denoted AtSCC4) was identified and shown to form functional complex with AtSCC2. Knockout of AtSCC4 induced inverse distribution of auxin response maxima in the embryos and ectopic cell division in the suspensor leading to developmental arrest and lethality. Split nuclei iFRAP (inverse fluorescence recovery after photobleaching) assay revealed AtSCC2-independent immobilization of AtSCC4 on chromatin and critical requirement of AtSCC4 for nuclear immobilization of cohesin.

During anaphase, sister chromatid cohesion is released by evolutionary conserved
protease separase (also called Extra Spindle Poles, ESP). Norway spruce (Picea abies) separase gene PaESP is highly expressed in the embryonal mass cells. PaESP-deficient embryos exhibited chromosome non-disjunction phenotype and perturbed anisotropic expansion of suspensor cells. Ectopic expression of PaESP could rescue chromosome non-disjunction phenotype of Arabidopsis ESP mutant (rsw4) but failed to rescue its root-swelling phenotype. This supports the notion of evolutionary conserved role of ESP in sister chromatid separation, but suggests that angiosperms and gymnosperms have evolved different molecular mechanisms of ESP-mediated regulation of cell expansion.

RNAseq analysis of transcriptomes of the Norway spruce embryo-suspensor
versus embryonal mass established a set of potential regulators of suspensor PCD. Most of these regulators are conserved across various plant lineages and have been implicated in the control of developmental PCD in Arabidopsis. Interestingly, the suspensor cells showed transcriptional up-regulation of a key component of endoplasmic reticulum stress (ER)-induced PCD, PaBI-1 (Picea abies Bax inhibitor-1), suggesting that suspensor PCD may likewise implicate ER-stress. Silencing of PaBI-1 induced necrotic cell death and abnormal suspensor phenotype leading to developmental arrest, thus establishing PaBI- 1 as an indispensable component of the suspensor PCD.