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Understanding the molecular mechanisms controlling the orientation of plant cell divisions

Periodic Reporting for period 4 - TORPEDO (Understanding the molecular mechanisms controlling the orientation of plant cell divisions)

Reporting period: 2021-08-01 to 2022-07-31

Context of the TORPEDO project:
Due to the presence of a cell wall, plant cells are fixed within their tissue context and cannot move relative to each other during development. Plants thus need to rely on directed cell elongation and cell division to generate a full three-dimensional (3D) structure. Intrinsic polarity cues and cellular communication provide spatial information to plant cells and establishes their position relative to the tissue context and the axis of growth. This framework allows cells to integrate available information and orient their divisions in such a way that structured growth becomes possible. Controlling cell division orientations relative to the tissue axis is therefore the fundamental basis for 3D growth. Plants utilize two main types of cell division to allow directional growth: anticlinal and periclinal cell divisions. Anticlinal cell divisions (AD) generate more cells within a certain cell file (perpendicular to the tissue axis) and are thus one of the main drivers for longitudinal growth in the mitotically active regions of the plant, called meristems. This division type is obviously not sufficient, as it would only generate filamentous structures. In order to create a 3D structure, plants use formative or periclinal divisions (PD) that generate additional cell files (parallel to the tissue axis). This results in radial growth and the formation of new organs. It is clear that a very precise control of cell division orientation is crucial to allow normal 3D growth to occur. Indeed, excessive activity of factors triggering PD in plants result in strong radial expansion in for example root tissues. This illustrates the need for cells to divide in a particular orientation at a precise moment in development. Therefore, understanding the mechanisms that control cell division orientation is a key question in developmental biology and the main focus of this application.

Impact of the TORPEDO project for society:
Vascular tissues undergo an enormous amount of PD as they grow from only a few cell files in the embryo into hundreds of cell files during secondary growth. This is very pronounced in trees, where PD generate almost all of the tissues in wood. Therefore, targeted engineering PD holds great potential for improving plant biomass and productivity to move towards a bio-based economy and thus reduce CO2 emissions countering global warming. Applications emerging from this project are not limited to these topics, but increased vascular tissue content in crop species can improve plant characteristics for producing bio-fuels or increase energy production by combustion of post-harvest waste.

Overall objectives of the TORPEDO project:
The main objective of this research proposal is to understand how plant cells control the orientation of their cell divisions, a process crucial for 3D growth in general and vascular development in specific. I aim to tackle this fundamental question in developmental biology by applying a set of distinct but complementary approaches.
1. As a first achievement of TORPEDO, we have identified mutants that are either resistant or hypersensitive to induction of PD; and have mapped the causal mutations to a set of novel genes. As these are very likely to be involved in this pathway, we will currently focus on understanding the function and localization of these genes. A manuscript describing these results is currently being revised for publication (bioRxiv 483486).
2. Besides this, as a second achievement, we have set-up a high throughput cell-based chemical genetics screening system to identify small molecules affecting oriented cell division, and have identified a set of 3 small molecules which are indeed affecting cell division orientation in both the cell-culture based screening system and in in planta experiments. We are focussing follow-up work to one chemical molecule able to induce oriented divisions in both our high-throughput cell culture system and in Arabidopsis roots. By analysing the transcriptional changes in these tissues upon treatment with this molecule, we have identified a transcription factor as top induced gene. Overexpression of this novel factor leads to a strong induction of oriented divisions in the root meristem, suggesting this factor is causal to the effect of the small molecule. We are currently following up on the biological role of this novel transcription factor in controlling cell division orientation as a main topic in the group.
3. We have additionally found the one of the main factors involved in controlling oriented cell divisions in the primary root meristem has a similar role throughout plant development. However, depending on the organ, different homologous transcription factors play a more prominent role. This means that heterodimer variations define oriented cell divisions in all plant meristem regions. These results are meanwhile published in iScience (bioRxiv 494582).
4. As a forth achievement in TORPEDO, we have identified a novel regulator of cell division orientation. Unlike the function of known pathways switching cell division orientation from AD to PD; this factor causes oblique divisions to occur. As such, we now have the perfect tools in hand to understand the difference between switching perfectly controlled oriented divisions and non-controlled oblique divisions.Work on this is currently being continued in the group.
5. As a final achievement of TORPEDO, we have set up a plant specific single cell RNA-sequencing pipeline in the host institute and are providing this to the plant community. Besides this, it has resulted in several high impact publications using this technology both from our group (Wendrich et al., 2020 Science; Yang et al., 2021 Nature Plants) and in collaboration with other labs (Graeff et al., 2021 Molecular Plant).

Work related to the TORPEDO project has been widely disseminated using alternative funding sources, including core funding:
- organization of 5 Vascular Development meetings in 2017-2022 (about 35-40 participants each)
- presentation using posters or oral presentations at several international meetings (including ROOTING2017, SEB2017, ACPD2018, iPMB2018, FASEB2019, CSHA2019, ROOTING2021, SEB2021, AUXIN2022 and others) and on invited lectures (including lectures in Helsinki, Paris-Saclay, Lyon, Nottingham, Tübingen, Zurich, Copenhagen, Lausanne and others). For a detailed overview, see the dissemination and output section.
Results at the end of the TORPEDO project:

We have made major advances in understanding how plant cells control the orientation of their cell divisions in the TORPEDO project (see above). This has been achieved on one hand by the identification of multiple novel regulators of oriented cell divisions in plants and a more holistic view of the function of the known regulators throughout plant development. On the other hand, we have developed and established novel technological platforms to continue research in this area, including a high-throughput plant cell culture based chemical screening system and a dedicated pipeline for single cell RNA-sequencing of plant samples. These will no doubt assist in further research unravelling the regulatory networks that control cell division orientation in plants.
The amount of active TMO5/LHW dimer controls vascular proliferation in a dose-dependent way