Dissection of cell division orientation control in Arabidopsis thaliana

Land plants can grow to exceptional body sizes, with the most complex specialized structures. Directional cell division has a fundamental role throughout the tremendous plant growth processes, yet its molecular regulation is still largely unknown. Chapter 1 of this thesis discusses mechanisms and sub-cellular structures known to be involved in plant cell division orientation control, and explores tools used for dissection of this complex mechanism.

Dissecting the complex plant cell division control mechanisms requires a simple and highly predictable in vivo model system. The highly predictable a relatively simple development of the Arabidopsis embryo makes it suitable for studying plant cell division regulation. In Chapter 2, we explore the cellular basis -, cell division patterns -, and regulatory pathways underlying early plant embryogenesis. We describe previous research showing that most cell divisions in the Arabidopsis embryo divide according a geometry- based “shortest-wall” principle, except for formative, asymmetric divisions. When auxin- signalling is disabled by overexpression of the dominant negative response inhibitor BODENLOS (BDL; bdl-mutant) all divisions in the embryo switch to “shortest-wall” divisions, suggesting auxin-signalling based control of oriented cell divisions.

Since the microtubule (MT) cytoskeleton is a possible factor regulating cell division orientation downstream of auxin, dissecting its dynamics and regulatory mechanisms is crucial to understand cell division regulation. Combining high-resolution imaging of cell walls and MTs with a modelling strategy for MT organisation, in Chapter 3, we show that the cortical MT array is crucial for division plane orientation control in de Arabidopsis embryo. In our model, MT-dynamics are confined by cell shape -, MT stability at cell edges-, and local MT stability by auxin. Additionally, comparison of cell-biology in wild-type - and bdl-mutant embryos in Chapter 4 reveals that F-actin organisation and cell expansion are affected in the mutant. These results suggest a function for the cytoskeleton and cell shape regulation in plant cell division orientation control downstream of BDL-mediated auxin-signalling.

Transcripts of factors regulating cytoskeletal structures and cell shape downstream of auxin should be differentially expressed in the bdl-mutant background. To allow for  the identification of these factors, in Chapter 5, we establish and optimize a pipeline to generate high-quality embryo transcriptomes for the earliest embryonic stages. As a proof of concept, we use this pipeline to generate a reference transcriptome for early Arabidopsis embryogenesis which can be used as a tool in future developmental embryonic research.

Since the role of auxin-signalling in cell division control is inferred from overexpression of a dominant response inhibitor, it is not clear if endogenous auxin truly regulates division orientation. In Chapter 6, we show that the segregating tir1/afb hexuple – and bdl-mutant embryos have similar division plane defects, suggesting that cell division regulation is regulated by auxin. However, we were not able to verify an overlap in transcriptomic regulation between the two auxin-insensitive mutants, possibly due to the small number of true mutants in the tir1/afb-mutant. Using the bdl-mutant transcriptome we identified IQ-domain 6 as a possible target of BDL-mediated auxin-signalling involved in cell division plane control. We show that the IQD6-8 protein family subclade localizes to MTs, and IQD6 binds cytoskeletal structures and calmodulins, in vivo. Loss of function of the IQD6-8 subclade results in skewed division planes in the embryo and roots, suggesting a role in cell division control, possible through Ca2+-mediated cytoskeletal regulation.

Finally, in Chapter 7, we discuss future challenges in research focused on plant cell division orientation control, and the most important findings of this thesis are placed in a broader cell biological – and plant developmental framework.