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Willemsen, V.

Publications and source records attributed to Willemsen, V..

12 recordsLinked to original sources

Diffusion-Based Synthetic Pretraining for Root Segmentation in Rhizotron Images

Rhizotrons allow root system architecture to be studied non-destructively in soil. However, automated analysis of rhizotron images remains difficult: roots have low contrast against soil, soil particles resemble fine roots, and deep learning models need many manually annotated images that are slow and subjective to produce. Synthetic images could provide paired images and annotations at scale, but rhizotron images are largely absent from the data used to train generative models. We built a pipeline combining Low-Rank Adaptation (LoRA), ControlNet and inpainting to generate synthetic rhizotron images with paired root masks. We compared training on synthetic data only, real data only, and synthetic pretraining followed by fine-tuning on real images, using three segmentation models: nnU-Net v2, YOLO26 and Mask2Former. Synthetic-only training reached a Dice score of up to 0.60. Synthetic pretraining followed by fine-tuning on the full real training set (two annotated rhizotron images) reached 0.80, and nnU-Net v2 and Mask2Former reached 0.62 with only two annotated image patches containing visible roots. When real annotations were limited, pretraining also improved segmentation of unseen crops, including lettuce, barley, wheat, maize, tomato, cucumber, French bean and field bean. We also introduced a Soft Dice score that compares predictions with probabilistic consensus maps from seven independent annotators, accounting for annotator disagreement. Models matched average human agreement on low-ambiguity patches but remained below it on high-ambiguity patches. In conclusion, diffusion-generated images can complement real annotations and help scale root segmentation, although their effect on downstream root trait measurements still needs to be validated.

plant biology↗

A complex balance: PLETHORA - JANUS ratiometry explains the PLETHORA dosage regeneration paradox

Molecular gradients are classically thought to encode positional information through concentration thresholds, but how transcription factor dosage is translated into regenerative competence remains unclear. In Arabidopsis roots, PLETHORA (PLT) transcription factors form a gradient that defines a regeneration competence zone and, following injury, rapidly re-establishes their transcriptional domain through an auto-activation. Yet, increasing PLT levels has a paradoxical effect: moderate increases expand the competence zone, whereas further increases impair regeneration. Here, combining mathematical modelling with experiments, we uncover the molecular basis of this non-monotonic response. Our model reveals that PLT activity depends not simply on its abundance, but on the balance between PLT protein complexes and unbound PLT. Following injury, PLT auto-activation rapidly restores the transcriptional domain, whereas increasing PLT dosage progressively shifts the equilibrium towards unbound PLT, reducing the availability of active PLT complexes. We identify and experimentally validate a strategy to extend regeneration competence by maintaining an appropriate ratio of PLT complexes to unbound PLT. Our findings reveal a ratiometric mechanism for decoding transcription factor dosage, in which excess regulator can attenuate rather than enhance activity. Such molecular balancing provides a mechanism by which a single transcription factor gradient can generate distinct, context-dependent outcomes during development and regeneration.

developmental biology↗

The MicroTron: a microfluidic platform for single cell studies in P. patens

O_LIThe effect of some bioactive compounds on living organisms is dependent on their concentration and gradients, as is the case of hormones and signalling peptides, determining cell identity, activity and organism development. C_LIO_LIThere are a handful of methods that allow to produce spatially confined peaks of concentration local application of biochemicals on plants, such as agar blocks and microinjection, but they lack in precision, throughput and/or simplicity. C_LIO_LIWe developed the MicroTron, a microfluidics-based method specifically for filamentous organisms or life cycle stages, like the moss plant Physcomitrium patens protonemata, that serves as a platform for the application of chemicals on single cells and study the cell response. C_LIO_LIWe show how chemical applications could be performed on cells, either on the side or apically with dyes and hormones, targeting the cell wall, cell membrane, cytosol and nucleus. C_LIO_LITreatments could be applied on single filaments and with a precision of up to single cells in optimal conditions. C_LIO_LIThis method could be used to study live responses to chemicals with high spatiotemporal resolution. C_LI

plant biology↗

A PLETHORA transcription factor shapes cucumber shoot architecture

O_LIPLETHORA transcription factors (PLTs) are master regulators of plant development. Loss of shoot meristematic PLTs leads to reduced phyllotactic regularity and robustness in Arabidopsis and increased inflorescence branching in tomato. Whether these factors have similar functions in other species is not known. C_LIO_LITo address this knowledge gap, we integrated phylogenetic, transcriptomic, and genome-wide in vitro binding strategies with quantitative shoot architecture phenotyping of a panel of cucumber TILLING mutants of each CsPLT homolog. C_LIO_LIWe determined that CsPLT3/7 and CsPLT are expressed in complementary domains in the cucumber shoot apex. DAP-seq data indicated that both transcription factors recognised an ANT-like consensus motif and bound to SAM organising genes. We identified strong phenotypic defects in multiple Csplt3/7 mutants. In mature seeds, Csplt3/7 mutants formed a flat apex instead of a shoot apical meristem, from which secondary meristems emerged during subsequent seedling development. Mature Csplt3/7 shoots exhibited defects in internode length regularity, stem architecture, flower morphology, and axillary organ initiation. Moreover, phyllotactic patterns were slightly shifted from a spiral towards a distichous orientation. C_LIO_LIWe present one of the first pieces of evidence that the PLT3/7 clade fulfils both conserved and diversified roles in shoot development across taxonomic family boundaries. C_LI

plant biology↗

Style deflection is determined by the handedness of phyllotaxis and differential cell elongation in a species with mirror-image flowers

Many animals and plants show left-right (LR) asymmetry. In some animal systems, handedness has a simple genetic basis, which has allowed identifying how handedness is determined at the molecular level, even if its functional relevance often remains unclear. Mirror-image flowers represent an example of LR asymmetry of clear functional significance, with the reciprocal placement of male and female organs in left- versus right-handed flowers promoting cross-pollination. Here, we use the South African geophyte Cyanella alba to study how handedness of its mirror-image flowers is determined and elaborated during development. Inflorescences of C. alba produce flowers with a largely consistent handedness. However, we find that this handedness has no simple genetic basis, and individual plants can switch their predominant handedness between years. Rather, it is the direction of the phyllotactic spiral that determines floral handedness. Cellular analysis combined with biophysical modelling demonstrates that style deflection is driven by increased cell expansion in the adaxial carpel facing the next oldest flower compared to the other adaxial carpel. The more expanding carpel shows transcriptional signatures of increased auxin signaling compared to the less expanding one, and auxin application to the latter can reverse the orientation of style deflection. We propose that a recently described inherent LR auxin asymmetry in the initiating organs of spiral phyllotaxis determines handedness in C. alba, representing a conserved non-genetic mechanism for creating a stable floral polymorphism. This mechanism links chirality across different levels of plant development and exploits a developmental constraint in a core patterning process to produce morphological variation of ecological relevance.

plant biology↗

Steep logarithmic increase of genetic variation in natural Arabidopsis thaliana accessions across geographic scales

Arabidopsis thalianas large native range across Eurasia and display of considerable genetic variation is key to its increasing use in eco-evolutionary studies. The structure and amount of this genetic variation has been studied on various geographic scales. On a continental scale, the genetic variation was postulated to follow an isolation by distance model, implying less genetic variation at smaller geographic distances. However, recent studies showed that the genetic variation is already high on small geographic scales, yet direct comparisons of the genetic variation across different geographic scales are rare. Here, we present a new local diversity panel covering 19 km2 with accessions of the Veluwe, the Netherlands. We compared the genetic variation of this local diversity panel to a national and a continental A. thaliana diversity panel. Direct comparison of these three geographic scales showed that local accessions harbour already 41.8% of the genetic variation found on a continental scale despite the substantial difference in geographic surface area covered. Moreover, a rapidly ascending logarithmic relationship between genetic and geographic distances was observed at continental, national and local scale and thus irrespective of the geographic scale considered. The high level of local genetic variation reported here poses new questions on which evolutionary forces are driving and maintaining this, and how much this constrains experimental design when using local A. thaliana populations in future eco-evolutionary studies.

genetics↗

WOX11-mediated cell size control in Arabidopsis attenuates fecundity of endoparasitic cyst nematodes

O_LICyst nematodes establish permanent feeding structures called syncytia inside host root vasculature, disrupting the flow of water and minerals. In response, plants form WOX11-mediated adventitious lateral roots at nematode infection sites. WOX11-adventitious lateral rooting modulates tolerance to nematode infections, however, whether this also benefits nematode parasitism remains unknown. C_LIO_LIHere, we report on bioassays using a 35S::WOX11-SRDX transcriptional repressor mutant to investigate whether WOX11-adventitious lateral rooting promotes syncytium development and thereby female fecundity. Moreover, we chemically inhibited cellulose biosynthesis to verify if WOX11 directly modulates cell wall plasticity in syncytia. Finally, we performed histochemical analyses to test if WOX11 mediates syncytial cell wall plasticity via reactive oxygen species (ROS). C_LIO_LIRepression of WOX11-mediated transcription specifically enhanced the radial expansion of syncytial elements, increasing both syncytium size and female offspring. The enhanced syncytial hypertrophy observed in the 35S::WOX11-SRDX mutant could be phenocopied by chemical inhibition of cellulose biosynthesis and was associated with elevated levels of ROS at nematode infection sites. C_LIO_LIWe therefore conclude that WOX11 restricts radial expansion of nematode feeding structures and female fecundity, likely by modulating ROS-mediated cell wall plasticity mechanisms. Remarkably, this novel role of WOX11 in plant cell size control is independent of WOX11-adventitious rooting underlying disease tolerance. C_LI

plant biology↗

CYP79B2 and CYP79B3 contribute to root branching through production of the auxin precursor indole-3-acetonitrile

Lateral root placement, outgrowth and density are influenced by environmental changes, including salinity stress. CYP79B2 and B3 are two cytochrome P450 enzymes previously identified as required for root architecture remodeling in salt. They produce iAOx, a metabolite that can be converted into indole glucosinolates (IGs), camalexin and indole-3-acetic acid (IAA), a type of auxin. We report here that lateral root appearance, induced by an auxin maximum in the bending zone after gravistimulation, is delayed in the absence of CYP79B2/B3. This delay traces back to a decrease in early lateral root growth after emergence, taking place before lateral roots are macroscopically visible. We measured gene transcripts and abundance of metabolites in the iAOx pathway in root segments that are forming lateral roots. Genes involved in tryptophane and IG biosynthesis were upregulated in cyp79b2/b3 mutants, suggesting a transcriptional feedback-loop. Salt stress was found to increase the expression of genes involved in IAN biosynthesis, a precursor of both IAA and camalexin, in the root during lateral root formation. Moreover, salt increases the concentration of IAN in tissue forming lateral roots in a CYP79B2/B3 dependent manner, but these changes in IAN did not coincide with altered IAA levels. Both the reduction in lateral root density under salt and the delayed lateral root appearance in cyp79b2/b3 knock-out mutants can be complemented by exogenous application of IAN. Our results reveal a role for the iAOx pathway in regulating the timing of lateral root appearance, allowing the modulation of lateral root density under salt stress.

plant biology↗

WOX11-mediated adventitious lateral root formation modulates tolerance of Arabidopsis to cyst nematode infections

The transcription factor WUSCHEL-RELATED HOMEOBOX 11 (WOX11) in Arabidopsis initiates the formation of adventitious lateral roots upon mechanical injury in primary roots. Root-invading nematodes also induce de novo root organogenesis leading to excessive root branching, but it is not known if this symptom of disease involves mediation by WOX11 and if it benefits the plant. Here, we show with targeted transcriptional repression and reporter gene analyses in Arabidopsis that the beet cyst nematode Heterodera schachtii activates WOX11-adventitious lateral rooting from primary roots close to infection sites. The activation of WOX11 in nematode-infected roots occurs downstream of jasmonic acid-dependent damage signaling via ETHYLENE RESPONSIVE FACTOR109, linking adventitious lateral root formation to nematode damage to host tissues. By measuring different root system components, we found that WOX11-mediated formation of adventitious lateral roots compensates for nematode-induced inhibition of primary root growth. Our observations further demonstrate that WOX11-mediated rooting reduces the impact of nematode infections on aboveground plant development and growth. Altogether, we conclude that the transcriptional regulation by WOX11 modulates root system plasticity under biotic stress, which is one of the key mechanisms underlying tolerance of Arabidopsis to cyst nematode infections.

plant biology↗

Root architecture plasticity in response to endoparasitic cyst nematodes is mediated by damage signaling

O_LIPlant root architecture plasticity in response to biotic stresses has not been thoroughly investigated. Infection by the endoparasitic cyst nematodes induces root architectural changes that involve the formation of secondary roots at infection sites. However, the molecular mechanisms regulating secondary root formation in response to cyst nematode infection remain largely unknown. C_LIO_LIWe first assessed whether secondary roots form in a nematode-density dependent manner by challenging wild type Arabidopsis plants with increasing numbers of cyst nematodes (Heterodera schachtii). Next, by using jasmonate-related reporter lines and knock-out mutants, we tested if tissue damage by nematodes triggers secondary root formation. Finally, we verified whether damage-induced secondary root formation depends on local auxin biosynthesis at nematode infection sites. C_LIO_LIIntracellular host invasion by H. schachtii triggers a transient local increase in jasmonates, which activates the expression of ERF109 in a COI1-dependent manner. Knock-out mutations in COI1 and ERF109 disrupt the nematode-density dependent increase of secondary roots observed in wildtype plants. Furthermore, ERF109 regulates secondary root formation upon H. schachtii infection via local auxin biosynthesis. C_LIO_LIHost invasion by H. schachtii triggers secondary root formation via the damage-induced jasmonate-dependent ERF109 pathway. This points at a novel mechanism underlying plant root plasticity in response to biotic stress. C_LI

plant biology↗

Redundant PLETHORA activity promotes development of early embryonic cell lineages in Arabidopsis

The BABY BOOM/PLETHORA4 (BBM/PLT4) transcription factor has received much attention due to its ability to induce somatic and zygotic embryogenesis, two processes of pivotal importance in plant breeding. Loss of additional AINTEGUMENTA-LIKE/PLETHORA (AIL/PLT) genes, encoding members of the APETALA2 transcription factor family, causes embryo arrest and abortion, but whether BBM/PLT4 provides specific information for embryo development has remained unknown. Here, we reveal that AIL/PLT members are expressed in partially overlapping domains from their first appearance in the apical cell daughter of the zygote. Redundant early embryonic activity of BABY BOOM/PLT4 and PLT2 triggers development of the apical cell lineage and is required to initiate embryonic primordia. Furthermore, promoter swap experiments show that PLT1 and PLT3 expression in the expression domains of PLT2 and BABY BOOM/PLT4 is sufficient to rescue plt2 bbm double mutants. Our data indicate that generic AIL/PLT factors, involved in maintenance of stem cells, promotion of cell division and suppression of cell differentiation, provide the necessary information to initiate embryogenesis in Arabidopsis.

plant biology↗

A microtubule-based mechanism predicts cell division orientation in plant embryogenesis

Oriented cell divisions are significant in plant morphogenesis because plant cells are embedded in cell walls and cannot relocate. Cell divisions follow various regular orientations, but the underlying mechanisms have not been clarified. We show that cell-shape dependent self-organisation of cortical microtubule arrays is crucial for determining planes of early tissue-generating divisions and forms the basis for robust control of cell division orientation in the embryo. To achieve this, we simulate microtubules on actual cell surface shapes from which we derive a minimal set of three rules for proper array orientation. The first rule captures the effects of cell shape alone on microtubule organisation, the second rule describes the regulation of microtubule stability at cell edges and the third rule includes the differential effect of auxin on local microtubule stability. These rules explain early embryonic division plane orientations and offer a framework for understanding patterned cell divisions in plant morphogenesis.

developmental biology↗