The Arabidopsis thaliana root is an excellent system to study oriented divisions, as there is clear spatial separation of morphologically distinct cell files along the main axis that are easy to follow throughout development. In particular the root tip, known as the root apical meristem, contains the stem cell niche and proliferation domains where new cells actively divide before reaching the elongation zone with their final cell fate. However, the plant body cannot achieve the necessary form only by cell divisions, especially since plant cells are immobile and have rigid cell walls. Thus, a key mechanism that shapes the plant is oriented divisions, of which there are two basic types: anticlinal and periclinal/radial. Anticlinal divisions are perpendicular to the  surface of the plant, while periclinal/radial divisions (PRDs) provide radial growth and are parallel to the surface. As such, anticlinal divisions add a cell to an existing cell file, while periclinal divisions generate a new cell file. In the root, PRDs are of particular developmental importance, as they are essential for formation of a fully patterned vascular system: centralized xylem pole, procambium and the phloem poles towards the outer edge. For example, in the phloem lineage, a procambium cell divides periclinally, resulting in a sieve element precursor cell, which then after some rounds of periclinal/radial divisions generates proto-phloem, meta-phloem, and companion cells. This illustrates how oriented divisions are essential for both vascular proliferation and the formation of new cell identities. Understanding the mechanism of cell division orientation in the root vasculature would thus shed light not only on one of the open fundamental questions in developmental and cell biology but also allow a deeper understanding of tissue establishment, which would then be translated to technologies that guide vascular proliferation in crops.

We have previously identified and described the TMO5/LHW-DOF2.1 transcriptional pathway as a key regulator of vascular proliferation via controlling oriented divisions, particularly through induction of PRDs. Mechanistically, in the xylem, the bHLH transcription factors TMO5 and LHW dimerize and bind to the promoter regions of the cytokinin biosynthesis genes, LOG3, LOG4 and BGLU44. This locally produced cytokinin acts as a mobile signal, travelling to the procambium cells and inducing DOF2.1, a transcription factor that initiates PRDs without induction of other cytokinin dependent events. Loss-of-function tmo5/lhw or dof higher order mutants show strong root vascular defects, while ectopic misexpression of TMO5/LHW or DOF2.1 shows an increase of PRDs, irrespective of cell and tissue identity, suggesting that these factors are enough to control PRDs. 

TMO5/LHW pathway

Figure: The TMO5/LHW pathway controls vascular cell proliferation and root hair responses to low phosphate conditions in the soil through local cytokinin biosynthesis and catabolism. Figure is based on the following publications from our group: Smet et al., Current Biology 2019; Wendrich et al., Science 2020; Yang et al., Nature Plants 2021; Mor, Pernisova et al., iScience 2022; Wybouw, Arents et al., J. Ex. Bot. 2023 and many great publications over the years from other research groups.

In summary, the TMO5/LHW and DOF2.1 transcription factors are part of the same molecular pathway driving oriented divisions in the root and can be used as tools to trigger these divisions and study the downstream events. In order to understand which players are involved downstream of the TMO5/LHW-DOF2.1 module, we have previously initiated forward and reverse screens, which have led to e.g. the identification of transcription factors involved in regulation of TMO5/LHW. However, no regulators bridging the upstream transcriptional responses and the downstream executors have so far been uncovered using these approaches, suggesting that these elusive regulators might be involved in non-transcriptional responses. We thus aim to unravel novel components guiding oriented divisions beyond the transcriptome.