Biology subjects

Nolf, J.

Publications and source records attributed to Nolf, J..

5 recordsLinked to original sources

bioRxiv2024

Shedding light on plant proteolysis: genetically encoded fluorescent sensors as tools for profiling protease activities

Proteolysis, a ubiquitous process in living organisms, is driven by proteases that regulate numerous signaling pathways through the hydrolysis of peptide bonds in protein substrates. Understanding the temporal and spatial dynamics of proteolysis and the activation of proteases is crucial for elucidating their roles in biological pathways. Here, we introduce a suite of genetically encoded FRET reporters designed to detect various proteolytic activities in plants. These sensors effectively reported in planta the specific activity of both Tobacco Etch Virus protease and caspase-3. Furthermore, we developed sensors for detecting plant metacaspase activity, validated through both in vitro and in planta experiments. These experiments revealed the spatial dynamics of proteolysis triggered by metacaspase activation following wounding and programmed cell death in roots. The implementation of these tools in plant biology research opens new avenues for investigating proteolytic mechanisms, significantly enhancing the potential for in-depth studies. Our work demonstrates the feasibility of using these sensors to detect diverse protease activities in vivo with high spatiotemporal resolution. These plant proteolytic biosensors hence represent a valuable toolbox for understanding protease functions within their natural context, paving the way for future advancements in plant biology research.

plant biology↗

bioRxiv2023

A precise balance of TETRASPANIN1/TORNADO2 activity is required for vascular proliferation and ground tissue patterning in Arabidopsis

The molecular mechanisms guiding oriented cell divisions in the root vascular tissues of Arabidopsis thaliana are still poorly characterized. By overlapping bulk and single-cell transcriptomic datasets, we unveiled TETRASPANIN1 (TET1) as a putative regulator in this process. TET1 is expressed in root vascular cells and loss-of-function mutants contain fewer vascular cells files. We further generated and characterized a CRISPR deletion mutant and show, unlike previously described mutants, that the full knock out is additionally missing endodermal cells in a stochastic way. Finally, we show that HA-tagged versions of TET1 are functional in contrast to fluorescent TET1 translational fusions. Immunostaining using HA-TET1 lines complementing the mutant phenotype revealed a dual plasma membrane and intracellular localization in the root vasculature and a polar membrane localization in young cortex, endodermal and initial cells. Taken together, we show that TET1 is involved in both vascular proliferation and ground tissue patterning. Our initial results pave the way for future work into deciphering its precise mode of action. Summary statementThis study reveals a novel role of tetraspanin TET1/TRN2 in root vascular development and ground tissue patterning in the model plant Arabidopsis thaliana.

plant biology↗

bioRxiv2022

bHLH heterodimer complex variations shape meristems in Arabidopsis thaliana by affecting target gene specificity

The main regions of cell proliferation in plants are the root and shoot apical meristems during primary growth and the vascular cambia as lateral meristems during secondary thickening. A number of unique regulators have been described in each of these meristems, suggesting that these different meristems might have independently evolved dedicated transcriptional networks to balance cell proliferation. Here, we show that the basic Helix Loop Helix (bHLH) transcription factor complexes formed by TARGET OF MONOPTEROS5 (TMO5), LONESOME HIGHWAY (LHW) and their close homologs are broadly expressed throughout plant development and operate as general regulators of cell proliferation in all meristems. Yet, genetic and expression analyses indicate that these complexes have specific functions in distinct meristems mediated by heterodimer complex variations between members of the TMO5 and LHW subclades. We determine that this is primarily due to their expression domains limiting the possible combinations of heterodimer complexes within a certain meristem, and to a certain extent to the absence of some members in a given meristem. We further demonstrate target gene specificity for heterodimer complexes, suggesting that spatial differences in transcriptional responses through heterodimer diversification allow a common bHLH heterodimer complex module to contribute to the control of cell proliferation in multiple meristems.

developmental biology↗

bioRxiv2022

MYB12 spatiotemporally represses TMO5/LHW-mediated transcription in the Arabidopsis root meristem

Transcriptional networks are crucial to integrate various internal and external signals into optimal responses during plant growth and development. Primary root vasculature patterning and proliferation are controlled by a network centred around the basic Helix-Loop-Helix transcription factor complex formed by TARGET OF MONOPTEROS 5 (TMO5) and LONESOME HIGHWAY (LHW), which control cell proliferation and orientation by modulating cytokinin response and other downstream factors. Despite recent progress, many aspects of the TMO5/LHW pathway are not fully understood. In particular, the upstream regulators of TMO5/LHW activity remain unknown. Here, using a forward genetic approach to identify new factors of the TMO5/LHW pathway, we discovered a novel function of the MYB-type transcription factor MYB12. MYB12 physically interacts with TMO5 and dampens the TMO5/LHW-mediated induction of direct target gene expression as well as the periclinal/radial cell divisions. The expression of MYB12 is activated by the cytokinin response, downstream of TMO5/LHW, resulting in a novel MYB12-mediated negative feedback loop that restricts TMO5/LHW activity to ensure optimal cell proliferation rates during root vascular development.

plant biology↗

bioRxiv2020

The TPLATE subunit is essential for structural assembly of the endocytic TSET complex

All eukaryotic cells rely on endocytosis to regulate the plasma membrane proteome and lipidome. Most eukaryotic groups, with the exception of fungi and animals, have retained the evolutionary ancient TSET complex as a regulator of endocytosis. Despite the presence of similar building blocks in TSET, compared to other coatomer complexes, structural insight into this adaptor complex is lacking. Here, we elucidate the molecular architecture of the octameric plant TSET complex (TPLATE complex/TPC) using an integrative structural approach. This allowed us to describe a plant-specific connection between the TML subunit and the AtEH/Pan1 proteins and show a direct interaction between the complex and the plasma membrane without the need for any additional protein factors. Furthermore, we identify the appendage of TPLATE as crucial for complex assembly. Structural elucidation of this ancient adaptor complex vastly advances our functional as well as evolutionary insight into the process of endocytosis. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

cell biology↗