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bioRxiv2025

Zinc deficiency induces spatially distinct responses in roots and impacts ZIP12-dependent zinc homeostasis in Arabidopsis

How zinc (Zn) deficiency shapes root development remains unclear, with conflicting reports on its effect on primary root growth in Arabidopsis thaliana (Arabidopsis). The impact of Zn shortage on the root apical meristem (RAM) in particular has not been systematically explored. Using an integrative approach combining cell biology, transcriptomics, and ionomics, we dissected how Zn deficiency alters root zonation and function. We showed that Zn deficiency triggers a striking reorganization of the root tip (RT): the RAM size is reduced, yet meristematic activity and local Zn levels are preserved. This is accompanied by promoted cell elongation and differentiation. Transcriptome profiling revealed a distinct Zn deficiency response in the RAM-enriched RT compared to mature root tissues, with ZIP12 emerging as the most strongly induced gene in the RT. Functional analysis of zip12 mutants uncovered major defects in root growth, RAM structure, expression of Zn-responsive genes, and metal partitioning. Our work unveiled a new layer of root developmental plasticity under Zn deficiency and identified ZIP12 as a central player in maintaining Zn homeostasis and root meristem function in Arabidopsis. These findings provide a framework to better understand how plants adapt root growth to fluctuating micronutrient availability.

plant biology↗

bioRxiv2025

Scaling law links plant growth variation to grain yield in wheat stands

Growth rate, a fundamental biological trait influencing plant resource use, scales predictably with plant mass, following remarkably consistent allometric power laws shaped by biophysical constraints and natural selection. However, how these laws apply to crop plants shaped by artificial selection, and how they manifest in agronomic traits, remains undefined. Under controlled greenhouse conditions, we quantified the relationship between plant mass and growth rate in 195 European winter wheat cultivars. We uncovered genetic variation in allometry linked to plant size, where increased leaf allocation and faster development elevated allometric exponents. Phenotypic and genetic analyses revealed adaptive strategies, ranging from large, slow-growing genotypes that support reproductive initiation to small, fast-growing genotypes that enhance reproductive effort. A shared genetic basis--associated with Photoperiod response-1 (Ppd-1)--linked growth allometry in the greenhouse to genotype-by-environment interactions for grain yield in field trials. This variation in growth allometry, shaped by breeding in diverse environments, reflects strategies that enhance adaptation to weather conditions. Our findings demonstrate that growth allometry is biologically robust and agronomically important because it scales to wheat yield under diverse, realistic field conditions.

plant biology↗

bioRxiv2021

Integrated analysis of the methylome and transcriptome of twin almonds (Prunus dulcis D.A.Webb) reveals genomic features associated with non-infectious bud failure

I.Almond (Prunus dulcis [Mill.] D.A.Webb) exhibits an age-related disorder called non-infectious bud-failure (BF) affecting vegetative bud development and nut yield. The underlying cause of BF remains unknown but is hypothesized to be associated with heritable epigenetic mechanisms. To address this disorder and its epigenetic components, we utilized a monozygotic twin study model profiling genome-wide DNA methylation and gene expression in two sets of twin almonds discordant for BF-exhibition. Analysis of DNA methylation patterns show that BF-exhibition and methylation, namely hypomethylation, are not independent phenomena. Transcriptomic data generated from the twin pairs also shows genome-wide differential gene expression associated with BF-exhibition. After identifying differentially methylated regions (DMRs) in each twin pair, a comparison revealed 170 shared DMRs between the two twin pairs. These DMRs and the associated genetic components may play a role in BF-exhibition. A subset of 52 shared DMRs are in close proximity to genes involved in meristem maintenance, cell cycle regulation, and response to heat stress. Annotation of specific genes included involvement in processes like cell wall development, calcium ion signaling, and DNA methylation. Results of this work support the hypothesis that BF-exhibition is associated with hypomethylation in almond, and identified DMRs and differentially expressed genes can serve as potential biomarkers to assess BF-potential in almond germplasm. Our results contribute to an understanding of the contribution of epigenetic disorders in agricultural performance and biological fitness of perennials. II. SignificanceThis study examines epigenetic components underlying noninfectious bud failure, an aging-related disorder affecting almond. Results from this work contribute to our understanding of the implications of DNA methylation on agricultural production, namely perennial fruit and nut production, due to effects on growth, development, and reproduction. Describing the methylome of discordant, monozygotic twin almonds enables the study of genomic features underlying noninfectious bud failure in this economically important crop.

plant biology↗

bioRxiv2023

A comparative analysis of stably expressed genes across diverse angiosperms exposes flexibility in underlying promoter architecture

Promoters regulate both the amplitude and pattern of gene expression--key factors needed for optimization of many synthetic biology applications. Previous work in Arabidopsis found that promoters that contain a TATA-box element tend to be expressed only under specific conditions or in particular tissues, while promoters which lack any known promoter elements, thus designated as Coreless, tend to be expressed more ubiquitously. To test whether this trend represents a conserved promoter design rule, we identified stably expressed genes across multiple angiosperm species using publicly available RNA-seq data. Comparisons between core promoter architectures and gene expression stability revealed differences in core promoter usage in monocots and eudicots. Furthermore, when tracing the evolution of a given promoter across species, we found that core promoter type was not a strong predictor of expression stability. Our analysis suggests that core promoter types are correlative rather than causative in promoter expression patterns and highlights the challenges in finding or building constitutive promoters that will work across diverse plant species.

plant biology↗

bioRxiv2022

Endogenous GFP tagging in the diatom Thalassiosira pseudonana

The regulated abundance and spatial distribution of proteins determines cellular structure and function. The discovery of green fluorescent protein (GFP) and fusing it to a target protein to determine subcellular localization revolutionized cell biology. Most localization studies involve introducing additional copies of a target gene genetically fused to GFP and under the control of a constitutive promoter, resulting in the expression of the GFP-fusion protein at non-native levels. Here we have developed a single vector CRISPR/Cas9 guided GFP knock-in strategy in the diatom Thalassiosira pseudonana. This enables precise and scarless knock-in of GFP at the endogenous genomic location to create GFP fusion proteins under their native cis and trans regulatory elements with knock-in efficiencies of over 50%. We show that a previously uncharacterized bestrophin-like protein localizes to the CO2-fixing pyrenoid and demonstrate that by measuring GFP fluorescence we can track relative protein abundance in response to environmental change. To enable endogenous tagging, we developed a Golden Gate Molecular Cloning system for the rapid assembly of episomes for transformation into Thalassiosira pseudonana via bacterial conjugation. In addition, this versatile toolbox enables CRISPR/Cas9 gene editing, provides a broad range of validated fluorophores and enables future large-scale functional studies in diatoms. Significance statementFluorescent protein (FP) tagging is a widely utilized technique for understanding the spatial distribution of proteins. However, introducing extra gene copies under constitutive promoters that randomly integrate into the genome can result in non-biologically relevant expression levels, unwanted genomic mutations and localization artefacts. To overcome this, we developed a novel single vector system capable of CRISPR/Cas9-guided endogenous GFP tagging in a globally important model diatom. This allows scarless GFP knock-in at precise genomic locations resulting in GFP fusions regulated by native promoters/terminators, which facilitates accurate localization and determination of relative protein abundance. Moreover, the developed modular cloning framework is user-friendly and opens the door for high throughput large-scale studies, including FP tagging, knock-out, and knock-in.

plant biology↗

bioRxiv2025

Benchmarking Phenotypic Clustering Algorithms via Empirically Calibrated Simulations: A Diagnostic Framework to Improve Biodiversity Assessment in Neglected Crops

Clustering algorithms are widely used for phenotypic characterization and germplasm management, particularly in neglected and underutilized species (NUS) that lack genomic resources. However, their performance under biologically realistic conditions remains poorly understood. Standard clustering methods commonly applied in crop research often assume distinct, isotropic, and homogeneous clusters--assumptions rarely satisfied in real-world NUS datasets. We developed a biologically informed simulation framework, empirically calibrated with phenotypic data from West African fonio (Digitaria exilis), to benchmark the performance of eleven clustering algorithms under both idealized and realistic scenarios. Our simulations integrated heterogeneous trait distributions (normal, gamma), strong inter-trait correlations (up to r = -0.84), heteroscedasticity, and moderate population structure (Pst {approx} 0.15), as observed in fonio landraces. Each scenario was replicated 100 times, with clustering accuracy evaluated using Adjusted Rand Index (ARI), Normalized Mutual Information (NMI), Silhouette coefficient, and Davies-Bouldin index. The results revealed consistently poor algorithm performance under realistic conditions (e.g., ARI < 0.07), including for widely used methods in NUS research such as K-means, GMM, and PAM. Performance markedly improved under idealized conditions, validating our simulation framework. These findings highlight the risk of overinterpreting clustering outputs from weakly structured phenotypic datasets and expose key limitations in current biodiversity analysis practices--particularly those guiding plant genetic resource conservation programs. We provide an open-source R-based diagnostic tool, available on Zenodo 5(https://doi.org/10.5281/zenodo.15877863), to assist practitioners in selecting robust clustering approaches for germplasm management and pre-breeding in data-scarce crops.

plant biology↗

bioRxiv2019

RNA G-quadruplex structures exist and function in vivo

Guanine-rich sequences are able to form complex RNA structures termed RNA G-quadruplexes in vitro. Because of their high stability, RNA G-quadruplexes are proposed to exist in vivo and are suggested to be associated with important biological relevance. However, there is a lack of direct evidence for RNA G-quadruplex formation in living cells. Therefore, it is unclear whether any purported functions are associated with the specific sequence content or the formation of an RNA G-quadruplex structure. Here, we profiled the landscape of those guanine-rich regions with the in vitro folding potential in the Arabidopsis transcriptome. We found a global enrichment of RNA G-quadruplexes with two G-quartets whereby the folding potential is strongly influenced by RNA secondary structures. Using in vitro and in vivo RNA chemical structure profiling, we determined that hundreds of RNA G-quadruplex structures are strongly folded in both Arabidopsis and rice, providing direct evidence of RNA G-quadruplex formation in living eukaryotic cells. Subsequent genetic and biochemical analysis showed that RNA G-quadruplex folding was sufficient to regulate translation and modulate plant growth. Our study reveals the existence of RNA G-quadruplex in vivo, and indicates that RNA G-quadruplex structures act as important regulators of plant development and growth.

plant biology↗

bioRxiv2021

An Arabidopsis root phloem pole cell atlas reveals PINEAPPLE genes as transitioners to autotrophy

Single cell sequencing has recently allowed the generation of exhaustive root cell atlases. However, some cell types are elusive and remain underrepresented. Here, we use a second- generation single cell approach, where we zoom in on the root transcriptome sorting with specific markers to profile the phloem poles at an unprecedented resolution. Our data highlight the similarities among the developmental trajectories and gene regulatory networks communal to protophloem sieve element (PSE) adjacent lineages in relation to PSE enucleation, a key event in phloem biology. As a signature for early PSE-adjacent lineages, we have identified a set of DNA-binding with one finger (DOF) transcription factors, the PINEAPPLEs (PAPL), that act downstream of PHLOEM EARLY DOF (PEAR) genes, and are important to guarantee a proper root nutrition in the transition to autotrophy. Our data provide a holistic view of the phloem poles that act as a functional unit in root development.

plant biology↗

bioRxiv2018

A phylogenetic framework of the legume genus Aeschynomene for comparative genetic analysis of the Nod-dependent and Nod-independent symbioses

SUMMARYO_LISome Aeschynomene legume species have the property of being nodulated by photosynthetic Bradyrhizobium lacking the nodABC genes. Knowledge of this unique Nod (factor)-independent symbiosis has been gained from the model A. evenia but our understanding remains limited due to the lack of comparative genetics with related taxa using a Nod-dependent process.\nC_LIO_LITo fill this gap, this study significantly broadened previous taxon sampling, including in allied genera, to construct a comprehensive phylogeny. This backbone tree was matched with data on chromosome number, genome size, low-copy nuclear genes and strengthened by nodulation tests and a comparison of the diploid species.\nC_LIO_LIThe phylogeny delineated five main lineages that all contained diploid species while polyploid groups were clustered in a polytomy and were found to originate from a single paleo-allopolyploid event. In addition, new nodulation behaviours were revealed and Nod-dependent diploid species were shown to be tractable.\nC_LIO_LIThe extended knowledge of the genetics and biology of the different lineages in the legume genus Aeschynomene provides a solid research framework. Notably, it enabled the identification of A. americana and A. patula as the most suitable species to undertake a comparative genetic study of the Nod-independent and Nod-dependent symbioses.\nC_LI

plant biology↗

bioRxiv2024

Callose Deficiency Modulates Plasmodesmata frequency and the intercellular space In Rice Anthers

Fertilization relies on pollen mother cells able to transit from mitosis-to-meiosis to supply gametes. This process involves remarkable changes at the molecular, cellular and physiological levels including (but not limited to) remodelling of cell wall. During meiosis onset, cellulose content at the pollen mother cell walls gradually declines with the concurrent deposition of the polysaccharide callose in anther locules. We aim to understand the biological significance of cellulose-to-callose turnover in pollen mother cells walls using electron microscopic analyses of rice flowers. Our observations indicate that in wild type anthers, the mitosis-to-meiosis transition coincides with a gradual reduction in the number of cytoplasmic connections called plasmodesmata. A mutant in the Oryza sativa callose synthase GSL5, impaired in callose accumulation in premeiotic and meiotic anthers, displayed a reduction in plasmodesmata frequency among pollen mother cells and tapetal cells suggesting a role for callose in plasmodesmata maintenance. In addition, a significant increase in cell-cell distance between pollen mother cells and impaired premeiotic cell shaping was observed in the mutant. The results suggest that cellulose-to-callose turnover during mitosis-meiosis transition is necessary to maintain cell-to-cell connections and optimal intercellular spacing among anther locular cells explaining the regulatory influence of callose metabolism during meiosis in flowering plants. HighlightsO_LICellulose-to-callose switch during meiosis onset in pollen mother cell walls correlates with changes in cytoplasmic connections called plasmodesmata, among meiocytes and between meiocytes and surrounding somatic cells. C_LIO_LIA mutant in GSL5 (glucan synthase like 5) affects callose deposition but no other cell wall component (e.g., cellulose) in premeiotic and meiotic anthers, except at dyad stage. C_LIO_LIImpaired callose synthesis is proposed to alter plasmodesmata frequency, the extracellular spacing and the shaping of pollen mother cells during mitosis-to-meiosis transition. C_LIO_LIIncrease in plasmodesmata frequency negatively correlates with cell-cell distance between pollen mother cells in both wildtype and a callose mutant. C_LI

plant biology↗

bioRxiv2020

Time-course transcriptome landscape of achene development in lettuce

Lettuce (Lactuca sativa L.), which belongs to the large Asteraceae (Compositae) family, breeds by sexual reproduction and produce seeds. Actually, lettuce seeds are achenes, which are defined as fruits. However, few studies have described the morphological characteristics of the lettuce achenes, and genes essential for achene development are largely unknown in lettuce. To investigate the gene activity during achene development and determine the possible mechanisms that influence achene development in lettuce, we performed a time-course transcriptome analysis of lettuce achenes. A total of 23,790 expressed genes were detected at the five achene development stages. We investigated the gene expression patterns during achene development and identified the enriched biological processes at the corresponding stages. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses revealed a variety of transcriptomic similarities and differentiation at different achene development stages. Further, transcription factors and phytohormones were found to play important roles during achene development. Finally, we proposed a working model to illustrate the gene expression modules and possible molecular mechanism underlying achene development. Our time-course transcriptome data also provides a foundation for future functional studies to reveal the genetic control of achene development in lettuce.

plant biology↗

bioRxiv2021

A Co-Fractionation Mass Spectrometry-based Prediction of Protein Complex Assemblies in the Developing Rice Aleurone-subaleurone

Multiprotein complexes execute and coordinate diverse cellular processes such as organelle biogenesis, vesicle trafficking, cell signaling, and metabolism. Knowledge about their composition and localization provides useful clues about the mechanisms of cellular homeostasis and systems-level control. This is of great biological importance and practical significance in heterotrophic rice endosperm and aleurone-subaleurone tissues that are a primary source of seed vitamins and stored energy. Dozens of protein complexes have been implicated in the synthesis, transport, and storage of seed proteins, lipids, vitamins, and minerals. Mutations in protein complexes that control RNA transport result in aberrant endosperm with shrunken and floury phenotypes, significantly reducing seed yield and quality. The purpose of this research is to broadly predict protein complex composition in the aleurone-subaleurone layers of developing rice seeds using co-fractionation mass spectrometry. Following orthogonal chromatographic separations of biological replicates, thousands of protein elution profiles were subjected to distance-based clustering to enable a large-scale determination of multimerization state and complex composition. Predictions included evolutionarily conserved proteins across diverse functional categories, including novel heteromeric RNA binding protein complexes that influence seed quality. This effective and open-ended proteomics pipeline provides useful clues about systems-level controls in the early stage of rice seed development. One-sentence summaryA co-fractionation mass spectrometry pipeline predicts compositions of cytosolic protein complexes present in the early stages of rice seed development.

plant biology↗

bioRxiv2022

Yield Prediction Through Integration of Genetic, Environment, and Management Data Through Deep Learning

Accurate prediction of the phenotypic outcomes produced by different combinations of genotypes, environments, and management interventions remains a key goal in biology with direct applications to agriculture, research, and conservation. The past decades have seen an expansion of new methods applied towards this goal. Here we predict maize yield using deep neural networks, compare the efficacy of two model development methods, and contextualize model performance using linear models, which are the conventional method for this task, and machine learning models We examine the usefulness of incorporating interactions between disparate data types. We find a deep learning model with interactions has the best average performance. Optimizing submodules for each datatype improved model performance relative to optimizing the whole model for all data types at once. Examining the effect of interactions in the best performing model revealed that including interactions altered the models sensitivity to weather and management features, including a reduction of the importance scores for timepoints expected to have limited physiological basis for influencing yield - those at the extreme end of the season, nearly 200 days post planting. Based on these results, deep learning provides a promising avenue for phenotypic prediction of complex traits in complex environments and a potential mechanism to better understand the influence of environmental and genetic factors.

plant biology↗

bioRxiv2024

Genetic and phenotypic characterization of global Lupinus albus genetic resources for the development of a CORE collection

Lupinus albus is a food grain legume recognized for its high levels of seed protein (30-40%) and oil (6-13%), and its adaptability to different climatic and soil conditions. To develop the next generation of L. albus cultivars, we need access to well-characterized, genetically and phenotypically diverse germplasm. Here we evaluated more than 2000 L. albus accessions with passport data based on 35 agro-morphological traits to develop Intelligent CORE Collections. The reference CORE (R-CORE), representing global diversity, exemplified the genotypic variation of cultivars, breeding/research materials, landraces and wild relatives. A subset of 300 R-CORE accessions was selected as a training CORE (T-CORE), representing the diversity in the entire collection. We divided the L. albus R-CORE into four phenotypic groups (A1, A2, A3 and B) based on principal component analysis, with groups A3 and B distinguished by pod shattering and seed ornamentation, respectively. The coefficient of additive genetic variation differed across morphological traits, phenotypic groups, geographic regions, and according to biological status. These CORE collections will facilitate agricultural research by identifying the genes responsible for desirable traits in crop improvement programs, and by shedding light on the use of orphan genetic resources for origin and domestication studies in L. albus. Understanding the variation in these genetic resources will allow us to develop sustainable tools and technologies that address global challenges such as providing healthy and sustainable diets for all, and contrasting the current climate change crisis.

plant biology↗

bioRxiv2021

Biological and Transcriptomic Characterization of Pre-haustorial Resistance to Sunflower Broomrape (Orobanche cumana W.)

Infestations with sunflower broomrape (Orobanche cumana Wallr.), an obligatory root parasite, constitute a major limitation to sunflower production in many regions around the world. Breeding for resistance is the most effective approach to reduce sunflower broomrape infestation, yet resistance mechanisms are often overcome by new races of the pathogen. Elucidating the mechanisms controlling the resistance to broomrape at the molecular level is thus the most desirable pathway to obtaining long-lasting resistance and reducing yield loss in sunflower. In this study, we investigated broomrape resistance in a confectionery sunflower hybrid with a robust and long-lasting resistance to sunflower broomrape. Visual screening and histological examination of sunflower roots revealed that penetration of the intrusive broomrape cells into the host root endodermis is blocked at the host cortex, indicating a pre-haustorial mechanism of resistance. A comparative RNA-Seq experiment conducted between roots obtained from the resistant cultivar, a bulk of five broomrape resistant lines and a bulk of five broomrape susceptible lines allowed the identification of genes that were significantly differentially expressed upon broomrape infestation. Among these differentially expressed genes, {beta}-1,3-endoglucanase, {beta}-glucanase and ethylene-responsive transcription factor4 (ERF4) genes were identified. These genes were previously reported to be pathogenesis-related genes in other plant species. This genetics investigation together with the histological examinations led us to conclude that the resistance mechanism involves the identification of the broomrape and the consequent formation of a physical barrier that prevents the penetration of the broomrape into the sunflower roots.

plant biology↗

bioRxiv2019

Custom built scanner and simple image processing pipeline enables low-cost, high-throughput phenotyping of maize ears

High-throughput phenotyping systems are becoming increasingly powerful, dramatically changing our ability to document, measure, and detect phenomena. Unfortunately, taking advantage of these trends can be difficult for scientists with few resources, particularly when studying nonstandard biological systems. Here, we describe a powerful, cost-effective combination of a custom-built imaging platform and open-source image processing pipeline. Our maize ear scanner was built with off-the-shelf parts for <$80. When combined with a cellphone or digital camera, videos of rotating maize ears were captured and digitally flattened into projections covering the entire surface of the ear. Segregating GFP and anthocyanin seed markers were clearly distinguishable in ear projections, allowing manual annotation using ImageJ. Using this method, statistically powerful transmission data can be collected for hundreds of maize ears, accelerating the phenotyping process.

plant biology↗

bioRxiv2026

A data-driven approach to integrative taxonomy

Central to understanding biodiversity is the systematic classification of biological taxa where concepts on integrating omics data have not yet been comprehensively executed. The data-driven approach is aiming at integrating phylogenetic data from three or more scales aiming at enriching the robustness of species delimitations while also revealing evolutionary drivers of diversification and speciation. This study investigates complex-thallose liverworts as reference and combines the analysis of DNA marker sequencing, morphometrics utilizing bioimaging measurements and chemometrics using liquid chromatography high-resolution mass-spectrometry (UPLC/ESI-QTOF-MS) with data-dependent acquisition of tandem mass-spectra (DDA-MS), with character variation evaluated with dendrograms and data mining. The comparative analysis revealed similar tree topologies albeit with diverging positions of taxa that were attributed to flavonoid-glycosides, auronidins and phenanthrenes resulting from evolutionary radiation and adaptations to stressful bioclimatic conditions, whereas unique fatty acyls in Riccia link to ecological causes of speciation. Characteristic amino acid motifs confirm the intermediary position of liverworts between algae and land plants. By functionally attributing and mapping chemometric markers to the taxonomy, the data-driven approach integrating omics into integrative taxonomy is opening opportunities for plant systematics to identify evolutionary drivers and form new hypotheses on the diversification and speciation of species.

plant biology↗

bioRxiv2021

Low cost, medium throughput depletion-binding assay for screening S-domain-receptor ligand interactions using in planta protein expression

BackgroundPlant cell-surface receptors sense various ligands to regulate physiological processes. Matching ligand-receptor pairs requires evidence of their direct interaction and is often a bottleneck in functional receptor studies. The S-domain-type (SD) pattern recognition receptor LORE senses medium chain-3-hydroxy fatty acids (mc-3-OH-FAs) such as 3-hydroxy decanoic acid (3-OH-C10:0) via its extracellular domain (ECD) They are perceived as signals of danger from Gram-negative bacteria and activate immune responses in Arabidopsis thaliana. LORE is found in low levels in planta and is poorly expressed in heterologous systems. Furthermore, chemical modifications of the mc-3-OH-FA ligand affect its biological activity. Taken together, this makes LORE-mc-3-OH-FA binding studies particularly challenging. ResultsTo investigate the LORE-mc-3-OH-FA interaction, we have developed a sensitive assay system based on protein expression in planta. The ECDs of LORE and other proteins of interest were transiently expressed as soluble, apoplastic mCherry fusion proteins in Nicotiana benthamiana and collected in apoplastic washing fluids. Protein-ligand complexes and unbound ligand were separated according to their molecular weight. In a two-step procedure, we first investigated whether the ECD-mCherry fusion protein depletes 3-OH-C10:0 from the low molecular weight fraction (step depletion). Subsequently, protein-bound 3-OH-C10:0 retained in the high molecular weight fraction in the depletion step is released and detected (step binding). Both the unbound and the released 3-OH-C10:0 ligand are detected by a sensitive bioassay using LORE loss- and gain-of-function Arabidopsis plants. Using the depletion-binding assay, we show that the ECD of AtLORE and its ortholog from Capsella rubella, CrubLORE, bind 3-OH-C10:0. The ECD of AtSD1-23, the closest paralog of AtLORE, and mCherry did not bind 3-OH-C10:0 and are suitable negative controls. ConclusionThe depletion-binding assay is a simple method for reliably detecting interactions between plant-expressed SD-type receptor ectodomains and mc-3-OH-FAs. It does not require special equipment or expensive consumables and is suitable for medium throughput screening. The assay is very flexible and can be easily adapted to investigate ligand interactions of other extracellular receptor domains.

plant biology↗