Search

Upon light-induced nuclear translocation, phytochrome (phy) sensory photoreceptors interact with, and induce rapid phosphorylation and consequent ubiquitin-mediated degradation of, transcription factors, called PIFs, thereby regulating target gene expression and plant development. Nevertheless, the biochemical mechanism of phy-induced PIF phosphorylation has remained ill-defined. Here we identify a family of nuclear protein kinases, designated Photoregulatory Protein Kinases (PPK1-4; formerly called MUT9-Like Kinases (MLKs)), that interact with PIF3 and phyB in a light-induced manner in vivo. Genetic analyses demonstrate that the PPKs are collectively necessary for the normal light-induced phosphorylation and degradation of PIF3. PPK1 directly phosphorylates PIF3 in vitro, with a phosphosite pattern that strongly mimics the light-induced pattern in vivo. These data establish that the PPKs are directly involved in catalysing the photoactivated-phy-induced phosphorylation of PIF3 in vivo, and thereby are critical components of a transcriptionally centred signalling hub that pleiotropically regulates plant growth and development in response to multiple signalling pathways.

Genetic studies have shown essential functions of O-linked N-acetylglucosamine (O-GlcNAc) modification in plants. However, the proteins and sites subject to this posttranslational modification are largely unknown. Here, we report a large-scale proteomic identification of O-GlcNAc-modified proteins and sites in the model plant Arabidopsis thaliana Using lectin weak affinity chromatography to enrich modified peptides, followed by mass spectrometry, we identified 971 O-GlcNAc-modified peptides belonging to 262 proteins. The modified proteins are involved in cellular regulatory processes, including transcription, translation, epigenetic gene regulation, and signal transduction. Many proteins have functions in developmental and physiological processes specific to plants, such as hormone responses and flower development. Mass spectrometric analysis of phosphopeptides from the same samples showed that a large number of peptides could be modified by either O-GlcNAcylation or phosphorylation, but cooccurrence of the two modifications in the same peptide molecule was rare. Our study generates a snapshot of the O-GlcNAc modification landscape in plants, indicating functions in many cellular regulation pathways and providing a powerful resource for further dissecting these functions at the molecular level.

After light-induced nuclear translocation, phytochrome photoreceptors interact with and induce rapid phosphorylation and degradation of basic helix-loop-helix transcription factors, such as PHYTOCHROME-INTERACTING FACTOR 3 (PIF3), to regulate gene expression. Concomitantly, this interaction triggers feedback reduction of phytochrome B (phyB) levels. Light-induced phosphorylation of PIF3 is necessary for the degradation of both proteins. We report that this PIF3 phosphorylation induces, and is necessary for, recruitment of LRB [Light-Response Bric-a-Brack/Tramtrack/Broad (BTB)] E3 ubiquitin ligases to the PIF3-phyB complex. The recruited LRBs promote concurrent polyubiqutination and degradation of both PIF3 and phyB in vivo. These data reveal a linked signal-transmission and attenuation mechanism involving mutually assured destruction of the receptor and its immediate signaling partner.

Plants adapt to environmental light conditions by photoreceptor-mediated physiological responses, but the mechanism by which photoreceptors perceive and transduce the signals is still unresolved. Here, we used 2D difference gel electrophoresis (2D DIGE) and mass spectrometry to characterize early molecular events induced by short blue light exposures in etiolated Arabidopsis seedlings. We observed the phosphorylation of phototropin 1 (phot1) and accumulation of weak chloroplast movement under blue light 1 (WEB1) in the membrane fraction after blue light irradiation. Over 50 spots could be observed for the two rows of phot1 spots in the 2-DE gels, and eight novel phosphorylated Ser/Thr sites were identified in the N-terminus and Hinge 1 regions of phot1 in vivo. Blue light caused ubiquitination of phot1, and K526 of phot1 was identified as a putative ubiquitination site. Our study indicates that post-translational modification of phot1 is more complex than previously reported.

Brassinosteroids (BRs) are essential phytohormones for plant growth and development. BRs are perceived by the cell surface receptor kinase BRI1, and downstream signal transduction through multiple components leads to activation of the transcription factors BZR1 and BZR2/BES1. BZR1 activity is highly controlled by BR through reversible phosphorylation, protein degradation, and nucleocytoplasmic shuttling. To further understand the molecular function of BZR1, we performed tandem affinity purification of the BZR1 complex and identified BZR1-associated proteins using mass spectrometry. These BZR1-associated proteins included several known BR signaling components, such as BIN2, BSK1, 14-3-3λ, and PP2A, as well as a large number of proteins with previously unknown functions in BR signal transduction, including the kinases MKK5 and MAPK4, histone deacetylase 19, cysteine proteinase inhibitor 6, a DEAD-box RNA helicase, cysteine endopeptidases RD21A and RD21B, calmodulin-binding transcription activator 5, ubiquitin protease 12, cyclophilin 59, and phospholipid-binding protein synaptotagmin A. Their interactions with BZR1 were confirmed by in vivo and in vitro assays. Furthermore, MKK5 was found to phosphorylate BZR1 in vitro. This study demonstrates an effective method for purifying proteins associated with low-abundance transcription factors, and identifies new BZR1-interacting proteins with potentially important roles in BR response.

When brassinosteroid levels are low, the GSK3-like kinase BIN2 phosphorylates and inactivates the BZR1 transcription factor to inhibit growth in plants. Brassinosteroid promotes growth by inducing dephosphorylation of BZR1, but the phosphatase that dephosphorylates BZR1 has remained unknown. Here, using tandem affinity purification, we identified protein phosphatase 2A (PP2A) as a BZR1-interacting protein. Genetic analyses demonstrated a positive role for PP2A in brassinosteroid signalling and BZR1 dephosphorylation. Members of the B' regulatory subunits of PP2A directly interact with BZR1's putative PEST domain containing the site of the bzr1-1D mutation. Interaction with and dephosphorylation by PP2A are enhanced by the bzr1-1D mutation, reduced by two intragenic bzr1-1D suppressor mutations, and abolished by deletion of the PEST domain. This study reveals a crucial function for PP2A in dephosphorylating and activating BZR1 and completes the set of core components of the brassinosteroid-signalling cascade from cell surface receptor kinase to gene regulation in the nucleus.

Sugar, light, and hormones are major signals regulating plant growth and development, however, the interactions among these signals are not fully understood at the molecular level. Recent studies showed that sugar promotes hypocotyl elongation by activating the brassinosteroid (BR) signaling pathway after shifting Arabidopsis seedlings from light to extended darkness. Here, we show that sugar inhibits BR signaling in Arabidopsis seedlings grown under light. BR induction of hypocotyl elongation in seedlings grown under light is inhibited by increasing concentration of sucrose. The sugar inhibition of BR response is correlated with decreased effect of BR on the dephosphorylation of BZR1, the master transcription factor of the BR signaling pathway. This sugar effect is independent of the sugar sensors Hexokinase 1 (HXK1) and Target of Rapamycin (TOR), but requires the GSK3-like kinase Brassinosteroid-Insensitive 2 (BIN2), which is stabilized by sugar. Our study uncovers an inhibitory effect of sugar on BR signaling in plants grown under light, in contrast to its promotive effect in the dark. Such light-dependent sugar-BR crosstalk apparently contributes to optimal growth responses to photosynthate availability according to light-dark conditions. Author summary: Genetic studies of the brassinosteroid (BR) deficient mutants revealed its essential role in seedling development in the dark, but subsequent studies showed no significant difference in BR level between seedlings grown under light and darkness. We recently observed that light does affect BR levels in Arabidopsis, but in a sugar dependent manner. In the dark, sugar increases BR level as well as BR sensitivity by stabilizing the steroid response factor BZR1 through the Target of Rapamycin (TOR) signaling pathway. However, the BR level is decreased by sugar under light and by darkness on sugar-free medium. These observations raised the question of how the combinations of light and sugar modulate BR signaling. We addressed this question using genetic physiological analyses and found interestingly that sugar inhibits brassinosteroid response in light-grown plants by stabilizing the glycogen synthase kinase 3 homolog BIN2 and attenuating the dephosphorylation of BZR1, but independently of TOR. Our results indicate that sugar acts through distinct pathways to promote and inhibit BR signaling in dark and light conditions. Our work illustrates an intricate three-way crosstalk whereby the combination of light and sugar signals modulate the brassinosteroid signaling pathway to optimize growth according to both environmental and metabolic conditions. [ABSTRACT FROM AUTHOR]

Plants must coordinate the regulation of biochemistry and anatomy to optimize photosynthesis and water-use efficiency. The formation of stomata, epidermal pores that facilitate gas exchange, is highly coordinated with other aspects of photosynthetic development. The signalling pathways controlling stomata development are not fully understood, although mitogen-activated protein kinase (MAPK) signalling is known to have key roles. Here we demonstrate in Arabidopsis that brassinosteroid regulates stomatal development by activating the MAPK kinase kinase (MAPKKK) YDA (also known as YODA). Genetic analyses indicate that receptor kinase-mediated brassinosteroid signalling inhibits stomatal development through the glycogen synthase kinase 3 (GSK3)-like kinase BIN2, and BIN2 acts upstream of YDA but downstream of the ERECTA family of receptor kinases. Complementary in vitro and in vivo assays show that BIN2 phosphorylates YDA to inhibit YDA phosphorylation of its substrate MKK4, and that activities of downstream MAPKs are reduced in brassinosteroid-deficient mutants but increased by treatment with either brassinosteroid or GSK3-kinase inhibitor. Our results indicate that brassinosteroid inhibits stomatal development by alleviating GSK3-mediated inhibition of this MAPK module, providing two key links; that of a plant MAPKKK to its upstream regulators and of brassinosteroid to a specific developmental output. [ABSTRACT FROM AUTHOR]

Brassinosteroids are essential hormones for plant growth and development. Genetic studies have identified key components of the BR signaling pathway, including the cell-surface receptor kinases that perceive BR, an intracellular kinase and a phosphatase, and nuclear transcription factors. Subsequent biochemical studies have revealed many details about signaling events from BR perception at the cell surface to gene expression in the nucleus. Recent studies have identified the 14-3-3 proteins as BR signaling components and elucidated multiple mechanisms by which phosphorylation modulates the BR transcription factors. In addition, BRI1 signaling from the endosomes and BR-independent functions of BAK1 have been observed. However, a major gap still exists in the current BR signaling pathway between the receptor complex and downstream signaling events. [Copyright &y& Elsevier]

Photomorphogenesis is a light-dependent plant growth and development program. As the core regulator of photomorphogenesis, ELONGATED HYPOCOTYL 5 (HY5) is affected by dynamic changes in its transcriptional activity and protein stability; however, little is known about the mediators of these processes. Here, we identified PHOTOREGULATORY PROTEIN KINASE 1 (PPK1), which interacts with and phosphorylates HY5 in Arabidopsis, as one such mediator. The phosphorylation of HY5 by PPK1 is essential to establish high-affinity binding with B-BOX PROTEIN 24 (BBX24) and CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), which inhibit the transcriptional activity and promote the degradation of HY5, respectively. As such, PPKs regulate not only the binding of HY5 to its target genes under light conditions but also HY5 degradation when plants are transferred from light to dark. Our data identify a PPK-mediated phospho-code on HY5 that integrates the molecular mechanisms underlying the regulation of HY5 to precisely control plant photomorphogenesis. [Display omitted] • PPKs regulate plant photomorphogenesis by phosphorylating HY5 • PPK1 phosphorylates HY5 at three residues: Thr55, Ser56, and Ser60 • Phosphorylation of HY5 by PPK1 enhances its interaction to COP1 • Phosphorylation of HY5 by PPK1 enhances its interaction to BBX24 In this study, Zhang and Wei et al. show that photoregulatory protein kinases (PPKs) interact with and phosphorylate HY5, thereby regulating its transcriptional activities and protein stability, which are essential for the precise control of plant photomorphogenesis. [ABSTRACT FROM AUTHOR]

Large numbers of receptor-like kinases (RLKs) play key roles in plant development and defense by perceiving extracellular signals. The mechanisms of ligand-induced kinase activation and downstream signal transduction have been studied for only a few RLK pathways, among which the brassinosteroid (BR) pathway is the best characterized. Recently, proteomics studies identified new components that bridge the last gap in the genetically defined BR-signaling pathway, establishing the first complete pathway from an RLK to transcription factors in plants. Furthermore, analyses of phosphorylation events, mostly by mass spectrometry, provided insights into the mechanistic details of receptor kinase activation and regulation of downstream components by phosphorylation. This review focuses on recent progress in understanding BR signal transduction made by proteomics studies. [Copyright &y& Elsevier]

Summary: The brassinosteroid (BR) signaling pathway includes two receptor-like kinases (BRI1 and BAK1), a plasma membrane-associated kinase (BSK1), two phosphatases (BSU1 and PP2A), a GSK3-like kinase (BIN2), and two homologous transcription factors (BZR1 and BES1/BZR2). But the mechanisms of signal relay are not fully understood. Here, we show that a receptor-like cytoplasmic kinase named CDG1 mediates signal transduction from BRI1 to BSU1. Transgenic experiments confirm that CDG1 and its homolog CDL1 positively regulate BR signaling and plant growth. Mass spectrometry analysis identified BRI1 phosphorylation sites in CDG1 and CDG1 phosphorylation sites in BSU1. Mutations of these phosphorylation sites compromised the BR signaling functions. The results demonstrate that BRI1 phosphorylates S234 to activate CDG1 kinase, and CDG1 in turn phosphorylates S764 to activate BSU1, which inactivates BIN2 by dephosphorylating Y200 of BIN2. This study thus demonstrates a complete phosphorylation/dephosphorylation cascade linking a steroid-activated receptor kinase to a GSK3-like kinase in plants. [ABSTRACT FROM AUTHOR]