Biology subjects

Broger, L.

Publications and source records attributed to Broger, L..

6 recordsLinked to original sources

bioRxiv2026

Plant Kelch phosphatases are Ser/Thr phosphatases involved in cell cycle regulation

Brassinosteroids (BRs) are plant steroid hormones sensed by the membrane receptor kinase BRI1. Activation of BRI1 leads to the dephosphorylation of BZR1/BES1 transcription factors. Overexpression of the Kelch phosphatase BRI1 SUPPRESSOR 1 (BSU1) rescued the growth defects of bri1 mutants. Subsequent studies identified BSU1 as a protein tyrosine phosphatase, which promotes BR signaling by dephosphorylating a phosphotyrosine in the glycogen synthase kinase 3 BIN2. Crystal structures of the BSU1 phosphatase domain now reveal a high degree of structural similarity to protein phosphatase 1 (PP1), a eukaryotic serine/threonine phosphatase. Consistently, BSU1 efficiently dephosphorylated phosphothreonine- and phosphoserine-containing substrate peptides, but showed no detectable activity toward BIN2 and other phosphotyrosine substrates. A catalytically inactive BSU1 phosphatase domain suppresses the growth phenotypes of the Arabidopsis bri1-5 mutant and binds the BSU1 homologs BSL1-3. bsu1 and bsu1 bsl1 bsl2/3 loss-of-function mutants display wild-type-like BR responses, but exhibit stomatal patterning and fertility defects. Importantly, the PP1-like C-terminal tail of BSU1 is phosphorylated at Thr785 by a cyclin-dependent kinase complex. The phosphorylated tail binds to the BSU1 substrate-binding grooves, blocking access to the active site. Mutation of Thr785 to alanine activates BSU1, suggesting that Kelch phosphatases and PP1 share a common regulatory mechanism. Deletion of the Marchantia polymorpha Kelch phosphatase MpBSLM results in an undifferentiated cell mass phenotype, associated with the over-activation of a cell cycle reporter. Taken together, our experiments suggest that plant Kelch phosphatases act as PP1-like cell cycle regulators, rather than as tyrosine phosphatases in brassinosteroid signaling. (243 words) Significance StatementBrassinosteroid hormones initiate a well-characterized signaling pathways in plants. While the early signaling events at the plasma membrane have been thoroughly examined, the precise sequence of molecular events that comprise the cytoplasmic signaling cascade is less well-understood. Here, the molecular function of the Kelch protein phosphatase BSU1 is re-evaluated. Previous reports have indicated that BSU1 dephosphorylates the GSK3 kinase BIN2 on a central tyrosine residue. However, structural, quantitative enzyme kinetic, and genetic analyses now suggest that BSU1 is a PP1-like serine/threonine phosphatase involved in cell cycle control rather than a tyrosine phosphatase involved in BIN2 de-phosphorylation. The regulation of BSU1 by a cyclin-dependent kinase complex and the role of Kelch phosphatases in cell cycle progression in Marchantia polymorpha are experimentally characterized. (121 words)

plant biology↗

bioRxiv2025

An inositol pyrophosphate interaction screen provides insight into the regulation of plant casein kinase II

Inositol pyrophosphates (PP-InsPs) are key nutrient messengers in plants, but their protein receptors remain poorly defined. Using a systems-level affinity screen with biotinylated InsP, InsP, and InsP in Arabidopsis thaliana, we identify multiple conserved PP-InsP-interacting complexes involved in mRNA metabolism, translation, and cell signaling, including the nuclear -subunits of casein kinase II (CK2). The CK2 subunit AtCKA1 associates with the PP-InsP kinase AtVIH2, and its 1.9 [A] crystal structure with InsP6 reveals two conserved PP-InsP binding sites located in the N-and C-terminal lobes. AtCKA1 binds InsP6, InsP7, and InsP8 with micromolar affinity. Mutation of both binding sites in the AtCKA6xmut mutant abolishes PP-InsP binding in vitro. AtCKA6xmut partially rescues the flowering phenotype of ck2a1/2/3 mutants, and equivalent mutations inactivate the yeast orthologs ScCka1 and ScCka2. InsP6 competitively inhibits phosphorylation of canonical CK2 substrates by occupying a basic substrate-binding groove. Although incorporating {beta}-subunits strongly enhances the phosphorylation of substrates by the AtCK2 holoenzyme, ck2b1/2/3/4 mutants exhibit only mild growth defects in Arabidopsis. In Marchantia, loss of the single ck2a gene severely impairs growth, whereas deletion of the {beta} subunit has no effect. Together, our findings suggest that InsP6/PP-InsPs modulate the activity of the isolated CK2 -subunit by regulating access to its substrate-binding site.

plant biology↗

bioRxiv2025

A mechanistic framework for the recognition of chemically diverse brassinosteroids by BRI1-family receptor kinases

Brassinosteroids (BRs) are chemically diverse plant steroid hormones produced via a branched biosynthetic pathway. The potent BR brassinolide is sensed by the membrane receptor kinase BRIl and a SERK co-receptor, but the physiological functions of other abundant BRs remain to be characterized. Here, we present quantitative binding kinetics for four Arabidopsis thaliana BR receptors and fifteen BRs, which define the key chemical features required for high affinity receptor binding, ligand positioning, and co-receptor recognition. BRIl, BRLl, and BRL3 share overlapping ligand preferences, whereas BRL2 preferentially binds C28 BRs with moderate affinity. Structural analyses of BR-bound BRIl and BRL3 ectodomains, combined with extensive in vitro and in vivo mutagenesis studies reveal a high structural plasticity of the hormone binding pocket. Functional assays using structure-based BR agonists and antagonists uncover that BR receptor - co-receptor signaling complexes can recognize chemically diverse BRs, introducing an additional, intriguing layer of BR signaling regulation.

plant biology↗

bioRxiv2019

Inositol pyrophosphates promote the interaction of SPX domains with the coiled-coil motif of PHR transcription factors to regulate plant phosphate homeostasis.

Phosphorus is an essential nutrient taken up by organisms in the form of inorganic phosphate (Pi). Eukaryotes have evolved sophisticated Pi sensing and signalling cascades, enabling them to maintain cellular Pi concentrations. Pi homeostasis is regulated by inositol pyrophosphate signalling molecules (PP-InsPs), which are sensed by SPX-domain containing proteins. In plants, PP-InsP bound SPX receptors inactivate Myb coiled-coil (MYB-CC) Pi starvation response transcription factors (PHRs) by an unknown mechanism. Here we report that a InsP8 - SPX complex targets the plant-unique CC domain of PHRs. Crystal structures of the CC domain reveal an unusual four-stranded anti-parallel arrangement. Interface mutations in the CC domain yield monomeric PHR1, which is no longer able to bind DNA with high affinity. Mutation of conserved basic residues located at the surface of the CC domain disrupt interaction with the SPX receptor in vitro and in planta, resulting in constitutive Pi starvation responses. Together, our findings suggest that InsP8 regulates plant Pi homeostasis by controlling the oligomeric state and hence the promoter binding capability of PHRs via their SPX receptors. (173 words)

plant biology↗

bioRxiv2019

A genetically validated approach to detect inorganic polyphosphates in plants.

Inorganic polyphosphates (polyPs) are linear polymers of orthophosphate units linked by phosphoanhydride bonds. PolyPs represent important stores of phosphate and energy, and are abundantly found in many pro- and eukaryotic organisms. In plants, the existence of polyPs has been established using microscopy and biochemical extraction methods that are now known to produce artifacts. Here we use a polyP-specific dye and a polyP binding domain to detect polyPs in plant and algal cells. To develop the staining protocol, we induced polyP granules in Nicotiana benthamiana and Arabiopsis cells by heterologous expression of E. coli polyphosphate kinase 1 (PPK1). Over-expression of PPK1 but not of a catalytically impaired version of the enzyme lead to severe growth phenotypes, suggesting that ATP-dependent synthesis and accumulation of polyPs in the plant cytosol is toxic. We next crossed stable PPK1 expressing Arabidopsis lines with plants expressing the polyP-binding domain of E. coli exopolyphosphatase (PPX1c), which co-localized with PPK1-generated polyP granules. These granules were stained by the polyP-specific dye JC-D7 and appeared as electron dense structures in transmission electron microscopy (TEM) sections. Using the polyP staining protocol derived from these experiments, we screened for polyP stores in different organs and tissues of both mono- and dicotyledonous plants. While we could not detect polyP granules in higher plants, we could visualize the polyP-rich acidocalicisomes in the green algae Chlamydomonas reinhardtii. Together, our experiments suggest that higher plants may not contain large polyPs stores.\n\nSignificance StatementA chemical dye and an inorganic polyphosphate binding domain are shown to specifically label inorganic polyphosphate granules in transgenic Arabidopsis lines and Chlamydomonas acidocalcisomes. Using these tools, we show that in contrast to many prokaryotic and eukaryotic organisms, higher plants do not seem to contain large inorganic polyphosphate stores.

plant biology↗

bioRxiv2018

Two bifunctional inositol pyrophosphate kinases/phosphatases control plant phosphate homeostasis.

Many eukaryotic proteins regulating phosphate (Pi) homeostasis contain SPX domains. We have previously shown that these domains act as cellular receptors for inositol pyrophosphate (PP-InsP) signaling molecules, suggesting that PP-InsPs may regulate Pi homeostasis. Here we report that simultaneous deletion of two diphosphoinositol pentakisphosphate kinases VIH1 and 2 in Arabidopsis impairs plant growth and leads to constitutive Pi starvation responses. We demonstrate that VIH1 and VIH2 are bifunctional cytosolic enzymes able to generate and break-down PP-InsPs. Point-mutants targeting the kinase and phosphatase active sites have opposing effects on plant Pi content and Pi starvation responses, while VIH1 and VIH2 protein levels remain constant in different Pi growth conditions. Enzymatic assays reveal that ATP-Mg2+ substrate levels can shift the relative kinase and phosphatase activities of full-length diphosphoinositol pentakisphosphate kinases. Deletion of phosphate starvation response transcription factors rescues vih1 vih2 mutant phenotypes, placing diphosphoinositol pentakisphosphate kinases and PP-InsPs in plant phosphate signal transduction cascades. We propose that VIH1 and VIH2 relay changes in cellular ATP concentration to changes in PPInsP levels, allowing plants to maintain cellular Pi concentrations constant and to trigger Pi starvation responses.

plant biology↗