OBERON3 and SUPPRESSOR OF MAX2 1-LIKE proteins form a regulatory module driving phloem development.

Spatial specificity of cell fate decisions is central for organismal development. The phloem tissue mediates long-distance transport of energy metabolites along plant bodies and is characterized by an exceptional degree of cellular specialization. How a phloem-specific developmental program is implemented is, however, unknown. Here we reveal that the ubiquitously expressed PHD-finger protein OBE3 forms a central module with the phloem-specific SMXL5 protein for establishing the phloem developmental program in Arabidopsis thaliana. By protein interaction studies and phloem-specific ATAC-seq analyses, we show that OBE3 and SMXL5 proteins form a complex in nuclei of phloem stem cells where they promote a phloem-specific chromatin profile. This profile allows expression of OPS, BRX, BAM3, and CVP2 genes acting as mediators of phloem differentiation. Our findings demonstrate that OBE3/SMXL5 protein complexes establish nuclear features essential for determining phloem cell fate and highlight how a combination of ubiquitous and local regulators generate specificity of developmental decisions in plants.

Three-Fluorophore FRET Enables the Analysis of Ternary Protein Association in Living Plant Cells.

Protein-protein interaction studies provide valuable insights into cellular signaling. Brassinosteroid (BR) signaling is initiated by the hormone-binding receptor Brassinosteroid Insensitive 1 (BRI1) and its co-receptor BRI1 Associated Kinase 1 (BAK1). BRI1 and BAK1 were shown to interact independently with the Receptor-Like Protein 44 (RLP44), which is implicated in BRI1/BAK1-dependent cell wall integrity perception. To demonstrate the proposed complex formation of BRI1, BAK1 and RLP44, we established three-fluorophore intensity-based spectral Förster resonance energy transfer (FRET) and FRET-fluorescence lifetime imaging microscopy (FLIM) for living plant cells. Our evidence indicates that RLP44, BRI1 and BAK1 form a ternary complex in a distinct plasma membrane nanodomain. In contrast, although the immune receptor Flagellin Sensing 2 (FLS2) also forms a heteromer with BAK1, the FLS2/BAK1 complexes are localized to other nanodomains. In conclusion, both three-fluorophore FRET approaches provide a feasible basis for studying the in vivo interaction and sub-compartmentalization of proteins in great detail.

Computational modeling and quantitative physiology reveal central parameters for brassinosteroid-regulated early cell physiological processes linked to elongation growth of the Arabidopsis root.

Brassinosteroids (BR) are key hormonal regulators of plant development. However, whereas the individual components of BR perception and signaling are well characterized experimentally, the question of how they can act and whether they are sufficient to carry out the critical function of cellular elongation remains open. Here, we combined computational modeling with quantitative cell physiology to understand the dynamics of the plasma membrane (PM)-localized BR response pathway during the initiation of cellular responses in the epidermis of the Arabidopsis root tip that are be linked to cell elongation. The model, consisting of ordinary differential equations, comprises the BR-induced hyperpolarization of the PM, the acidification of the apoplast and subsequent cell wall swelling. We demonstrate that the competence of the root epidermal cells for the BR response predominantly depends on the amount and activity of H+-ATPases in the PM. The model further predicts that an influx of cations is required to compensate for the shift of positive charges caused by the apoplastic acidification. A potassium channel was subsequently identified and experimentally characterized, fulfilling this function. Thus, we established the landscape of components and parameters for physiological processes potentially linked to cell elongation, a central process in plant development.

A data management infrastructure for the integration of imaging and omics data in life sciences.

BACKGROUND: As technical developments in omics and biomedical imaging increase the throughput of data generation in life sciences, the need for information systems capable of managing heterogeneous digital assets is increasing. In particular, systems supporting the findability, accessibility, interoperability, and reusability (FAIR) principles of scientific data management. RESULTS: We propose a Service Oriented Architecture approach for integrated management and analysis of multi-omics and biomedical imaging data. Our architecture introduces an image management system into a FAIR-supporting, web-based platform for omics data management. Interoperable metadata models and middleware components implement the required data management operations. The resulting architecture allows for FAIR management of omics and imaging data, facilitating metadata queries from software applications. The applicability of the proposed architecture is demonstrated using two technical proofs of concept and a use case, aimed at molecular plant biology and clinical liver cancer research, which integrate various imaging and omics modalities. CONCLUSIONS: We describe a data management architecture for integrated, FAIR-supporting management of omics and biomedical imaging data, and exemplify its applicability for basic biology research and clinical studies. We anticipate that FAIR data management systems for multi-modal data repositories will play a pivotal role in data-driven research, including studies which leverage advanced machine learning methods, as the joint analysis of omics and imaging data, in conjunction with phenotypic metadata, becomes not only desirable but necessary to derive novel insights into biological processes.

The EDS1-PAD4-ADR1 node mediates Arabidopsis pattern-triggered immunity.

Plants deploy cell-surface and intracellular leucine rich-repeat domain (LRR) immune receptors to detect pathogens1. LRR receptor kinases and LRR receptor proteins at the plasma membrane recognize microorganism-derived molecules to elicit pattern-triggered immunity (PTI), whereas nucleotide-binding LRR proteins detect microbial effectors inside cells to confer effector-triggered immunity (ETI). Although PTI and ETI are initiated in different host cell compartments, they rely on the transcriptional activation of similar sets of genes2, suggesting pathway convergence upstream of nuclear events. Here we report that PTI triggered by the Arabidopsis LRR receptor protein RLP23 requires signalling-competent dimers of the lipase-like proteins EDS1 and PAD4, and of ADR1 family helper nucleotide-binding LRRs, which are all components of ETI. The cell-surface LRR receptor kinase SOBIR1 links RLP23 with EDS1, PAD4 and ADR1 proteins, suggesting the formation of supramolecular complexes containing PTI receptors and transducers at the inner side of the plasma membrane. We detected similar evolutionary patterns in LRR receptor protein and nucleotide-binding LRR genes across Arabidopsis accessions; overall higher levels of variation in LRR receptor proteins than in LRR receptor kinases are consistent with distinct roles of these two receptor families in plant immunity. We propose that the EDS1-PAD4-ADR1 node is a convergence point for defence signalling cascades, activated by both surface-resident and intracellular LRR receptors, in conferring pathogen immunity.

Specifying the role of BAK1-interacting receptor-like kinase 3 in brassinosteroid signaling.

Brassinosteroids (BR) are involved in the control of several developmental processes ranging from root elongation to senescence and adaptation to environmental cues. Thus, BR perception and signaling have to be precisely regulated. One regulator is BRI1-associated kinase 1 (BAK1)-interacting receptor-like kinase 3 (BIR3). In the absence of BR, BIR3 forms complexes with BR insensitive 1 (BRI1) and BAK1. However, the biophysical and energetic requirements for complex formation in the absence of the ligand have yet to be determined. Using computational modeling, we simulated the potential complexes between the cytoplasmic domains of BAK1, BRI1 and BIR3. Our calculations and experimental data confirm the interaction of BIR3 with BAK1 and BRI1, with the BAK1 BIR3 interaction clearly favored. Furthermore, we demonstrate that BIR3 and BRI1 share the same interaction site with BAK1. This suggests a competition between BIR3 and BRI1 for binding to BAK1, which results in preferential binding of BIR3 to BAK1 in the absence of the ligand thereby preventing the active participation of BAK1 in BR signaling. Our model also suggests that BAK1 and BRI1 can interact even while BAK1 is in complex with BIR3 at an additional binding site of BAK1 that does not allow active BR signaling.

A Mutant Allele Uncouples the Brassinosteroid-Dependent and Independent Functions of BRASSINOSTEROID INSENSITIVE 1.

Plants depend on various cell surface receptors to integrate extracellular signals with developmental programs. One of the best-studied receptors is BRASSINOSTEROID INSENSITIVE 1 (BRI1) in Arabidopsis (Arabidopsis thaliana). Upon binding of its hormone ligands, BRI1 forms a complex with a shape-complementary coreceptor and initiates a signal transduction cascade, which leads to a variety of responses. At the macroscopic level, brassinosteroid (BR) biosynthetic and receptor mutants have similar growth defects, which initially led to the assumption that the signaling pathways were largely linear. However, recent evidence suggests that BR signaling is interconnected with several other pathways through various mechanisms. We recently described that feedback from the cell wall is integrated at the level of the receptor complex through interaction with RECEPTOR-LIKE PROTEIN 44 (RLP44). Moreover, BRI1 is required for another function of RLP44: the control of procambial cell fate. Here, we report a BRI1 mutant, bri1 cnu4 , which differentially affects canonical BR signaling and RLP44 function in the vasculature. Although BR signaling is only mildly impaired, bri1 cnu4 mutants show ectopic xylem in place of procambium. Mechanistically, this is explained by an increased association between RLP44 and the mutated BRI1 protein, which prevents the former from acting in vascular cell fate maintenance. Consistent with this, the mild BR response phenotype of bri1 cnu4 is a recessive trait, whereas the RLP44-mediated xylem phenotype is semidominant. Our results highlight the complexity of plant plasma membrane receptor function and provide a tool to dissect BR signaling-related roles of BRI1 from its noncanonical functions.

Targeted manipulation of bZIP53 DNA-binding properties influences Arabidopsis metabolism and growth.

bZIP transcription factors regulate diverse processes in eukaryotic cells. Arabidopsis bZIP members of the C and S1 groups form heterodimers and synergistically control metabolic reprogramming during stress responses. However, their functional characterization is complicated due to an overlapping heterodimerization network and high redundancy. In this study, we develop a simple but powerful approach for generating dominant negative mutants of bZIP factors with high specificity. By applying in vitro DNA-binding, reporter gene and protoplast two-hybrid assays, and plant mutant analysis, we show that phosphorylation-mimicking substitution of conserved serines in the DNA-binding domain of bZIP monomeric subunits suffices for the disruption of the interaction of both bZIP homo- and heterodimers with cognate DNA. This results in the transcriptional inactivation of target genes. The dominant-negative effect is achieved by the unaltered function of the intrinsic nuclear localization signal and dimerization properties of the mutated bZIP protein. Our findings not only reveal an additional regulatory mechanism of bZIP10 intracellular localization, but also provide evidence of the involvement of bZIP53 in the diurnal adjustments of amino acid metabolism. Our data demonstrate the advantages and the suitability of this new approach for the artificial inactivation of bZIP transcription factors in plants, and it may also be of use for other organisms.

BRI1 controls vascular cell fate in the Arabidopsis root through RLP44 and phytosulfokine signaling.

Multicellularity arose independently in plants and animals, but invariably requires a robust determination and maintenance of cell fate that is adaptive to the environment. This is exemplified by the highly specialized water- and nutrient-conducting cells of the plant vasculature, the organization of which is already prepatterned close to the stem-cell niche, but can be modified according to extrinsic cues. Here, we show that the hormone receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) is required for root vascular cell-fate maintenance, as BRI1 mutants show ectopic xylem in procambial position. However, this phenotype seems unrelated to canonical brassinosteroid signaling outputs. Instead, BRI1 is required for the expression and function of its interacting partner RECEPTOR-LIKE PROTEIN 44 (RLP44), which, in turn, associates with the receptor for the peptide hormone phytosulfokine (PSK). We show that PSK signaling is required for the maintenance of procambial cell identity and quantitatively controlled by RLP44, which promotes complex formation between the PSK receptor and its coreceptor. Mimicking the loss of RLP44, PSK-related mutants show ectopic xylem in the position of the procambium, whereas rlp44 is rescued by exogenous PSK. Based on these findings, we propose that RLP44 controls cell fate by connecting BRI1 and PSK signaling, providing a mechanistic framework for the dynamic balancing of signaling mediated by the plethora of plant receptor-like kinases at the plasma membrane.