SignificanceCell-fate determination and cellular behavior in plants rely mainly on positional information and intercellular communication. A plethora of cues are perceived by surface receptors and integrated into an adequate cellular output. Here, we show that the small receptor-like protein RLP44 acts as an intermediary to connect the receptors for two well-known signaling molecules, brassinosteroid and phytosulfokine, to control cell fate in the root vasculature. Furthermore, we show that the brassinosteroid receptor has functions that are independent from the responses to its hormone ligands and reveal that phytosulfokine signaling promotes procambial cell identity. These results provide a mechanistic framework for the integration of multiple signaling pathways at the plasma membrane by shifting associations of receptors in multiprotein complexes.

Plants have evolved complex photoreceptor-controlled mechanisms to sense and respond to seasonal changes in day length. This ability allows plants to optimally time the transition from vegetative growth to flowering. UV-B is an important part intrinsic to sunlight; however, whether and how it affects photoperiodic flowering has remained elusive. Here, we report that, in the presence of UV-B, genetic mutation of REPRESSOR OF UV-B PHOTOMOR-PHOGENESIS 2 (RUP2) renders the facultative long day plant Arabidopsis thaliana a day-neutral plant and that this phenotype is dependent on the UV RESISTANCE LOCUS 8 (UVR8) UV-B photoreceptor. We provide evidence that the floral repression activity of RUP2 involves direct interaction with CONSTANS, repression of this key activator of flowering, and suppression of FLOWERING LOCUS T transcription. RUP2 therefore functions as an essential repressor of UVR8-mediated induction of flowering under noninductive short day conditions and thus provides a crucial mechanism of photoperiodic flowering control.

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Plants depend on various cell surface receptors to integrate extracellular signals with developmental programs. One of the beststudied 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. Plant cells perceive a multitude of extracellular signals through a battery of plasma membrane-bound receptors that are crucial for the integration of environmental and developmental signals. The response to the growth-regulatory brassinosteroid (BR) phytohormones is mediated by one of the best-characterized plant signaling pathways (Singh and Savaldi-Goldstein, 2015) initiated by a receptor complex containing the Leu-rich repeat receptor-like kinase BRASSINOSTE-ROID INSENSITIVE 1 (BRI1; Li and Chory, 1997) and its coreceptors of the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family (Ma et al., 2016; Hohmann et al., 2017). Binding of the brassinosteroid ligand mediates hetero-dimerization of BRI1 and SERK family members such as BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1; Li et al., 2002; Nam and Li, 2002), which in turn triggers extensive autoand transphosphorylation of the intracellular BAK1 and BRI1 kinase domains (Hohmann et al., 2017). The activated kinases recruit and activate downstream BR signaling components, which eventually leads to vast changes in gene expression mediated by BR signalingregulated transcription factors such as BRASSINA-ZOLE-RESISTANT 1 (BZR1; Wang et al., 2002) and BRI1-EMS-SUPPRESSOR 1 (BES1; Yin et al., 2002). Among the transcriptional targets of these transcription factors, cell...

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.

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.

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Integration of signalling on the cellular level is essential for the survival of organisms.Protein-protein interaction studies provide valuable insights in these signalling events.One of the best understood signalling pathways in plants is the brassinosteroid (BR) hormone signalling pathway, which is mediated by the receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) with its co-receptor BRI1-ASSOCIATED KINASE (BAK1). Both BRI1 and BAK1 have been shown to interact with RECEPTOR LIKE PROTEIN 44 (RLP44), which was implicated in cell wall integrity sensing by modulation of BL signalling. Here we provide evidence by quantitative in vivo three-fluorophore FRET-FLIM measurements, that RLP44, BRI1 and BAK1 form a trimeric complex in the plasma membrane of N. benthamiana leaf cells, with an estimated distance between them below 15 nm. The immune receptor FLAGELLIN SENSING 2 (FLS2), which is also a receptor-like kinase like BRI1, is not integrated in a similar complex with RLP44 and BAK1. Our study supports that BRI1 and FLS2 are localized in distinct nanodomains in the PM. As the fluorescence lifetime of the donor is monitored, our method circumvents the extensive calculations necessitated by intensity-based FRET interaction assays and thus provides a feasible base for studying the sub-compartmentalization in the plasma membrane of living plant cells with a nanoscale resolution.