Tyrosine phosphorylation controls brassinosteroid receptor activation by triggering membrane release of its kinase inhibitor

Receptor tyrosine kinases control many critical processes in metazoans, but these enzymes appear to be absent in plants. Recently, two Arabidopsis receptor kinases-BRASSINOSTEROID INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED KINASE1 (BAK1), the receptor and coreceptor for brassinosteroids-were shown to autophosphorylate on tyrosines. However, the cellular roles for tyrosine phosphorylation in plants remain poorly understood. Here, we report that the BRI1 KINASE INHIBITOR 1 (BKI1) is tyrosine phosphorylated in response to brassinosteroid perception. Phosphorylation occurs within a reiterated [KR][KR] membrane targeting motif, releasing BKI1 into the cytosol and enabling formation of an active signaling complex. Our work reveals that tyrosine phosphorylation is a conserved mechanism controlling protein localization in all higher organisms

ttl mutants are impaired in cellulose biosynthesis under osmotic stress

As sessile organisms, plants require mechanisms to sense and respond to the challenging environment, that encompass both biotic and abiotic factors that results in differential development. In these conditions is essential to balance growth and stress responses. As cell walls shape plant growth, this differential growth response cause alterations to the plant cell wall and cellulose is a major component. Therefore, understanding the mechanisms that regulate cellulose biosynthesis is essential to develop strategies to improve plant production. Previous studies have shown that the GSK3 kinase BIN2 modulate cellulose biosynthesis through phosphorylating cellulose synthases and that the expression of cellulose synthases are regulated by Brassinosteroids. Our previous work reveals that the tetratricopeptide-repeat thioreoxin-like (TTL) TTL1, TTL3, and TTL4 genes, in addition to their reported role in abiotic stress tolerance, are positive regulators of BR signaling. We observe association of TTL3 with most core components in traducing BR signalling, such as LRR-RLK BRI1, BIN2 and the transcription factor BES1 that positively regulate cellulose biosynthesis. We show that ttl mutants are affected in cellulose biosynthesis, particularly in osmotic stress conditions. Furthermore, TTL3 associates with LRR-RLKs that have been shown to be important for cellulose biosynthesis such as FEI1 in the FEI1/FEI2/SOS5 pathway. We aim to investigate the mechanisms by which TTL proteins regulate cellulose biosynthesis using a combination of genetics, biochemical, and molecular and cell biology approaches. This work was supported by grants from: (1) Ministerio de Ciencia e Innovación BIO2014-55380-R, BIO2014-56153-REDT; (2) Ministerio de Economía, Industria y Competitividad (BES-2015-071256); (3) Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.This work was supported by grants from: (1) Ministerio de Ciencia e Innovación BIO2014-55380-R, BIO2014-56153-REDT; (2) Ministerio de Economía, Industria y Competitividad (BES-2015-071256); (3) Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Unraveling the mechanism of TTL genes in cellulose biosynthesis

As sessile organisms, plants require mechanisms to sense and respond to the challenging environment, that encompass both biotic and abiotic factors that results in differential development. In these conditions is essential to balance growth and stress responses. As cell walls shape plant growth, this differential growth response cause alterations to the plant cell wall where cellulose is the major component. Therefore, understanding the mechanisms that regulate cellulose biosynthesis is essential to develop strategies to improve plant production. In Arabidopsis, the TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) gene family is composed by four members (TTL1 to TTL4) and mutations in TTL1, TTL3, and TTL4 genes cause reduced growth under salt and osmotic stress due to defects in plant cell wall integrity. We observe association of TTL3 with most core components in traducing BR signalling, such as LRR-RLK BRI1 or GSK3 BIN2 that modulate cellulose biosynthesis through phosphorylating cellulose synthases. Here, we show that ttl mutants present defects in the plant cell wall, particularly in Isoxaben, salt or sucrose stress. Spinning disk microscopy in etiolated hypocotyls reveals that, TTL proteins are responsible for the cellulose synthase complex (CSC) stability in plasma membrane (PM) upon sucrose stress. Moreover, TTL3 associates with LRR-RLKs that have been shown to be important for cellulose biosynthesis such as FEI1 in the FEI1/FEI2/SOS5 pathway. We aim to investigate the mechanisms by which TTL proteins regulate CesA stability in PM under stress, using a combination of genetics, biochemical, and molecular and cell biology approaches.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This work was supported by grants from: (1) Ministerio de Ciencia e Innovación BIO2014-55380-R, BIO2014-56153-REDT; (2) Ministerio de Economía, Industria y Competitividad (BES-2015-071256

The Arabidopsis translocator protein (AtTSPO) is regulated at multiple levels in response to salt stress and perturbations in tetrapyrrole metabolism

<p>Abstract</p> <p>Background</p> <p>The translocator protein 18 kDa (TSPO), previously known as the peripheral-type benzodiazepine receptor (PBR), is important for many cellular functions in mammals and bacteria, such as steroid biosynthesis, cellular respiration, cell proliferation, apoptosis, immunomodulation, transport of porphyrins and anions. <it>Arabidopsis thaliana </it>contains a single <it>TSPO/PBR</it>-related gene with a 40 amino acid N-terminal extension compared to its homologs in bacteria or mammals suggesting it might be chloroplast or mitochondrial localized.</p> <p>Results</p> <p>To test if the TSPO N-terminal extension targets it to organelles, we fused three potential translational start sites in the <it>TSPO </it>cDNA to the N-terminus of GFP (<it>At</it>TSPO:eGFP). The location of the <it>At</it>TSPO:eGFP fusion protein was found to depend on the translational start position and the conditions under which plants were grown. Full-length <it>At</it>TSPO:eGFP fusion protein was found in the endoplasmic reticulum and in vesicles of unknown identity when plants were grown in standard conditions. However, full length <it>At</it>TSPO:eGFP localized to chloroplasts when grown in the presence of 150 mM NaCl, conditions of salt stress. In contrast, when <it>At</it>TSPO:eGFP was truncated to the second or third start codon at amino acid position 21 or 42, the fusion protein co-localized with a mitochondrial marker in standard conditions. Using promoter <it>GUS </it>fusions, qRT-PCR, fluorescent protein tagging, and chloroplast fractionation approaches, we demonstrate that <it>At</it>TSPO levels are regulated at the transcriptional, post-transcriptional and post-translational levels in response to abiotic stress conditions. Salt-responsive genes are increased in a <it>tspo-1 knock-down </it>mutant compared to wild type under conditions of salt stress, while they are decreased when <it>At</it>TSPO is overexpressed. Mutations in tetrapyrrole biosynthesis genes and the application of chlorophyll or carotenoid biosynthesis inhibitors also affect <it>AtTSPO </it>expression.</p> <p>Conclusion</p> <p>Our data suggest that AtTSPO plays a role in the response of <it>Arabidopsis </it>to high salt stress. Salt stress leads to re-localization of the AtTSPO from the ER to chloroplasts through its N-terminal extension. In addition, our results show that <it>AtTSPO </it>is regulated at the transcriptional level in tetrapyrrole biosynthetic mutants. Thus, we propose that <it>At</it>TSPO may play a role in transporting tetrapyrrole intermediates during salt stress and other conditions in which tetrapyrrole metabolism is compromised.</p

Dual role for ubiquitin in plant steroid hormone receptor endocytosis

Brassinosteroids are plant steroid hormones that control many aspects of plant growth and development, and are perceived at the cell surface by the plasma membrane-localized receptor kinase BRI1. Here we show that BRI1 is post-translationally modified by K63 polyubiquitin chains in vivo. Using both artificial ubiquitination of BRI1 and generation of an ubiquitination-defective BRI1 mutant form, we demonstrate that ubiquitination promotes BRI1 internalization from the cell surface and is essential for its recognition at the trans-Golgi network/early endosomes (TGN/EE) for vacuolar targeting. Finally, we demonstrate that the control of BRI1 protein dynamics by ubiquitination is an important control mechanism for brassinosteroid responses in plants. Altogether, our results identify ubiquitination and K63-linked polyubiquitin chain formation as a dual targeting signal for BRI1 internalization and sorting along the endocytic pathway, and highlight its role in hormonally controlled plant development

SEC14-like condensate phase transitions at plasma membranes regulate root growth in Arabidopsis

Protein function can be modulated by phase transitions in their material properties, which can range from liquid- to solid-like; yet, the mechanisms that drive these transitions and whether they are important for physiology are still unknown. In the model plant Arabidopsis, we show that developmental robustness is reinforced by phase transitions of the plasma membrane-bound lipid-binding protein SEC14-like. Using imaging, genetics, and in vitro reconstitution experiments, we show that SEC14-like undergoes liquid-like phase separation in the root stem cells. Outside the stem cell niche, SEC14-like associates with the caspase-like protease separase and conserved microtubule motors at unique polar plasma membrane interfaces. In these interfaces, SEC14-like undergoes processing by separase, which promotes its liquid-to-solid transition. This transition is important for root development, as lines expressing an uncleavable SEC14-like variant or mutants of separase and associated microtubule motors show similar developmental phenotypes. Furthermore, the processed and solidified but not the liquid form of SEC14-like interacts with and regulates the polarity of the auxin efflux carrier PINFORMED2. This work demonstrates that robust development can involve liquid-to-solid transitions mediated by proteolysis at unique plasma membrane interfaces.The mechanisms that drive protein phase transitions are unclear. This study in plants shows that an intracellular liquid condensate formed by the lipid transferase SFH8 associates with membranes; when a short fragment of SFH8 is removed by the caspase-like protease ESP, it transforms into a solid filament that can modulate root development

The receptor kinase FERONIA regulates phosphatidylserine localization at the cell surface to modulate ROP signaling

Cells maintain a constant dialog between the extracellular matrix and their plasma membrane to fine tune signal transduction processes. We found that the receptor kinase FERONIA (FER), which is a proposed cell wall sensor, modulates phosphatidylserine plasma membrane accumulation and nano-organization, a key regulator of Rho GTPase signaling in Arabidopsis. We demonstrate that FER is required for both Rho-of-Plant 6 (ROP6) nano-partitioning at the membrane and downstream production of reactive oxygen species upon hyperosmotic stimulus. Genetic and pharmacological rescue experiments indicate that phosphatidylserine is required for a subset of, but not all, FER functions. Furthermore, application of FER ligand shows that its signaling controls both phosphatidylserine membrane localization and nanodomains formation, which, in turn, tunes ROP6 signaling. Together, we propose that a cell wall-sensing pathway controls via the regulation of membrane phospholipid content, the nano-organization of the plasma membrane, which is an essential cell acclimation to environmental perturbations

SEC14-like condensate phase transitions at plasma membranes regulate root growth in Arabidopsis.

Protein function can be modulated by phase transitions in their material properties, which can range from liquid- to solid-like; yet, the mechanisms that drive these transitions and whether they are important for physiology are still unknown. In the model plant Arabidopsis, we show that developmental robustness is reinforced by phase transitions of the plasma membrane-bound lipid-binding protein SEC14-like. Using imaging, genetics, and in vitro reconstitution experiments, we show that SEC14-like undergoes liquid-like phase separation in the root stem cells. Outside the stem cell niche, SEC14-like associates with the caspase-like protease separase and conserved microtubule motors at unique polar plasma membrane interfaces. In these interfaces, SEC14-like undergoes processing by separase, which promotes its liquid-to-solid transition. This transition is important for root development, as lines expressing an uncleavable SEC14-like variant or mutants of separase and associated microtubule motors show similar developmental phenotypes. Furthermore, the processed and solidified but not the liquid form of SEC14-like interacts with and regulates the polarity of the auxin efflux carrier PINFORMED2. This work demonstrates that robust development can involve liquid-to-solid transitions mediated by proteolysis at unique plasma membrane interfaces

SAUR63 stimulates cell growth at the plasma membrane

In plants, regulated cell expansion determines organ size and shape. Several members of the family of redundantly acting Small Auxin Up RNA (SAUR) proteins can stimulate plasma membrane (PM) H+-ATPase proton pumping activity by inhibiting PM-associated PP2C.D phosphatases, thereby increasing the PM electrochemical potential, acidifying the apoplast, and stimulating cell expansion. Similarly, Arabidopsis thaliana SAUR63 was able to increase growth of various organs, antagonize PP2C.D5 phosphatase, and increase H+-ATPase activity. Using a gain-of-function approach to bypass genetic redundancy, we dissected structural requirements for SAUR63 growth-promoting activity. The divergent N-terminal domain of SAUR63 has a predicted basic amphipathic α-helix and was able to drive partial PM association. Deletion of the N-terminal domain decreased PM association of a SAUR63 fusion protein, as well as decreasing protein level and eliminating growth-promoting activity. Conversely, forced PM association restored ability to promote H+-ATPase activity and cell expansion, indicating that SAUR63 is active when PM-associated. Lipid binding assays and perturbations of PM lipid composition indicate that the N-terminal domain can interact with PM anionic lipids. Mutations in the conserved SAUR domain also reduced PM association in root cells. Thus, both the N-terminal domain and the SAUR domain may cooperatively mediate the SAUR63 PM association required to promote growth