Isabelle Vanhoutte

SPEECHLESS integrates brassinosteroid and stomata signalling pathways

Stomatal formation is regulated by multiple developmental and environmental signals, but how these signals are integrated to control this process is not fully understood. In Arabidopsis thaliana, the basic helix-loop-helix transcription factor SPEECHLESS (SPCH) regulates the entry, amplifying and spacing divisions that occur during stomatal lineage development. SPCH activity is negatively regulated by mitogen-activated protein kinase (MAPK)-mediated phosphorylation. Here, we show that in addition to MAPKs, SPCH activity is also modulated by brassinosteroid (BR) signalling. The GSK3/SHAGGY-like kinase BIN2 (BR INSENSITIVE2) phosphorylates residues overlapping those targeted by the MAPKs, as well as four residues in the amino-terminal region of the protein outside the MAPK target domain. These phosphorylation events antagonize SPCH activity and limit epidermal cell proliferation. Conversely, inhibition of BIN2 activity in vivo stabilizes SPCH and triggers excessive stomatal and non-stomatal cell formation. We demonstrate that through phosphorylation inputs from both MAPKs and BIN2, SPCH serves as an integration node for stomata and BR signalling pathways to control stomatal development in Arabidopsis.

Chemical Inhibition of a Subset of Arabidopsis thaliana GSK3-like Kinases Activates Brassinosteroid Signaling

Glycogen synthase kinase 3 (GSK3) is a key regulator in signaling pathways in both animals and plants. Three Arabidopsis thaliana GSK3s are shown to be related to brassinosteroid (BR) signaling. In a phenotype-based compound screen we identified bikinin, a small molecule that activates BR signaling downstream of the BR receptor. Bikinin directly binds the GSK3 BIN2 and acts as an ATP competitor. Furthermore, bikinin inhibits the activity of six other Arabidopsis GSK3s. Genome-wide transcript analyses demonstrate that simultaneous inhibition of seven GSK3s is sufficient to activate BR responses. Our data suggest that GSK3 inhibition is the sole activation mode of BR signaling and argues against GSK3-independent BR responses in Arabidopsis. The opportunity to generate multiple and conditional knockouts in key regulators in the BR signaling pathway by bikinin represents a useful tool to further unravel regulatory mechanisms.

A New Class of Ubiquitin Extension Proteins Secreted by the Dorsal Pharyngeal Gland in Plant Parasitic Cyst Nematodes

By performing cDNA AFLP on pre- and early parasitic juveniles, we identified genes encoding a novel type of ubiquitin extension proteins secreted by the dorsal pharyngeal gland in the cyst nematode Heterodera schachtii. The proteins consist of three domains, a signal peptide for secretion, a mono-ubiquitin domain, and a short C-terminal positively charged domain. A gfp-fusion of this protein is targeted to the nucleolus in tobacco BY-2 cells. We hypothesize that the C-terminal peptide might have a regulatory function during syncytium formation in plant roots.

The clathrin adaptor complex AP-2 mediates endocytosis of brassinosteroid insensitive1 in Arabidopsis.

In mammals, clathrin-mediated endocytosis (CME) depends on the heterotetrameric ADAPTOR PROTEIN COMPLEX-2 (AP-2). Our work identifies the components of the Arabidopsis thaliana AP-2 and shows that the machinery of CME in plants is evolutionarily conserved. Our data reveal that AP-2 mediates the endocytosis of the brassinosteroid receptor BRI1. Clathrin-mediated endocytosis (CME) regulates many aspects of plant development, including hormone signaling and responses to environmental stresses. Despite the importance of this process, the machinery that regulates CME in plants is largely unknown. In mammals, the heterotetrameric ADAPTOR PROTEIN COMPLEX-2 (AP-2) is required for the formation of clathrin-coated vesicles at the plasma membrane (PM). Although the existence of AP-2 has been predicted in Arabidopsis thaliana, the biochemistry and functionality of the complex is still uncharacterized. Here, we identified all the subunits of the Arabidopsis AP-2 by tandem affinity purification and found that one of the large AP-2 subunits, AP2A1, localized at the PM and interacted with clathrin. Furthermore, endocytosis of the leucine-rich repeat receptor kinase, BRASSINOSTEROID INSENSITIVE1 (BRI1), was shown to depend on AP-2. Knockdown of the two Arabidopsis AP2A genes or overexpression of a dominant-negative version of the medium AP-2 subunit, AP2M, impaired BRI1 endocytosis and enhanced the brassinosteroid signaling. Our data reveal that the CME machinery in Arabidopsis is evolutionarily conserved and that AP-2 functions in receptor-mediated endocytosis.

Brassinosteroid production and signaling differentially control cell division and expansion in the leaf

Brassinosteroid (BR) hormones control plant growth through acting on both cell expansion and division. Here, we examined the role of BRs in leaf growth using the Arabidopsis BR-deficient mutant constitutive photomorphogenesis and dwarfism (cpd). We show that the reduced size of cpd leaf blades is a result of a decrease in cell size and number, as well as in venation length and complexity. Kinematic growth analysis and tissue-specific marker gene expression revealed that the leaf phenotype of cpd is associated with a prolonged cell division phase and delayed differentiation. cpd-leaf-rescue experiments and leaf growth analysis of BR biosynthesis and signaling gain-of-function mutants showed that BR production and BR receptor-dependent signaling differentially control the balance between cell division and expansion in the leaf. Investigation of cell cycle markers in leaves of cpd revealed the accumulation of mitotic proteins independent of transcription. This correlated with an increase in cyclin-dependent kinase activity, suggesting a role for BRs in control of mitosis.

An SXP/RAL-2 protein produced by the subventral pharyngeal glands in the plant parasitic root-knot nematode Meloidogyne incognita

Meloidogyne incognita is a major parasite of numerous plant families, including many crop species. Upon infection of the plant root, it induces several multinucleate giant cells by the injection of pharyngeal gland secretions into the root cells. In order to obtain a better understanding of the nematode-plant interaction, characterization of the pharyngeal gland secretions is a necessity. By differential display, a nematode gene was identified that encodes a new member of the SXP/RAL-2 protein family. The gene is specifically expressed in the subventral pharyngeal glands and the protein is most likely secreted.

Helix-loop-helix/basic helix-loop-helix transcription factor network represses cell elongation in Arabidopsis through an apparent incoherent feed-forward loop

Significance Our work provides an insight into the complex network of basic helix–loop–helix (bHLH)/helix–loop–helix (HLH) transcription factors that regulates cell elongation. An unknown network motif (an incoherent feed-forward loop) has been discovered that was established by two negative regulators of brassinosteroid responses, namely the HLH transcription factor INCREASED LEAF INCLINATION1 BINDING bHLH1 (IBH1) and its unidentified homolog, IBH1-LIKE1 (IBL1). We also suggest that IBH1 and IBL1 coexist with PHYTOCHROME-INTERACTING FACTOR 4 (another key regulator of cell elongation) in transcriptional complexes. Cell elongation is promoted by different environmental and hormonal signals, involving light, temperature, brassinosteroid (BR), and gibberellin, that inhibit the atypical basic helix–loop–helix (bHLH) transcription factor INCREASED LEAF INCLINATION1 BINDING bHLH1 (IBH1). Ectopic accumulation of IBH1 causes a severe dwarf phenotype, but the cell elongation suppression mechanism is still not well understood. Here, we identified a close homolog of IBH1, IBH1-LIKE1 (IBL1), that also antagonized BR responses and cell elongation. Genome-wide expression analyses showed that IBH1 and IBL1 act interdependently downstream of the BRASSINAZOLE-RESISTANT1 (BZR1)–PHYTOCHROME-INTERACTING FACTOR 4 (PIF4)–DELLA module. Although characterized as non-DNA binding, IBH1 repressed direct IBL1 transcription, and they both acted in tandem to suppress the expression of a common downstream helix–loop–helix (HLH)/bHLH network, thus forming an incoherent feed-forward loop. IBH1 and IBL1 together repressed the expression of PIF4, known to stimulate skotomorphogenesis synergistically with BZR1. Strikingly, PIF4 bound all direct and down-regulated HLH/bHLH targets of IBH1 and IBL1. Additional genome-wide comparisons suggested a model in which IBH1 antagonized PIF4 but not the PIF4–BZR1 dimer.

An improved method for whole-mount in situ hybridization of Heterodera schachtii juveniles

Abstract. An optimized protocol is presented to visualize gene expression in the sedentary beet cyst nematode, Heterodera schachtii, by whole-mount in situ hybridization. Two different probes were used for genes with known expression pattern in other nematodes. Vacuum infiltration of the fixative significantly increased its efficiency and resulted in a nicely preserved morphology. Additional modifications were introduced to simplify and standardize the process.

Regulation of Arabidopsis brassinosteroid receptor BRI1 endocytosis and degradation by plant U-box PUB12/PUB13-mediated ubiquitination

Significance The brassinosteroid (BR) receptor BRI1 provides a paradigm for understanding receptor-mediated signaling in plants. Different posttranslational modifications have been implicated in the regulation of BRI1 activity. Here, we show that BR perception promotes BRI1 association with plant U-box E3 ubiquitin ligases PUB12 and PUB13, which in turn directly ubiquitinate BRI1. Importantly, the BRI1 protein abundance and plasma membrane-residence time are increased while the endosomal pool of BRI1 is reduced in the pub12pub13 mutant, indicating that PUB12/PUB13-mediated ubiquitination regulates BRI1 endocytosis and degradation. BRI1 phosphorylates PUB13 on a specific residue to enhance its association with BRI1, suggesting a unique regulatory circuit of phosphorylation-regulated E3 ligase–substrate association. Our study elucidates a mechanism of BRI1 internalization through E3 ubiquitin ligase-mediated ubiquitination. Plants largely rely on plasma membrane (PM)-resident receptor-like kinases (RLKs) to sense extracellular and intracellular stimuli and coordinate cell differentiation, growth, and immunity. Several RLKs have been shown to undergo internalization through the endocytic pathway with a poorly understood mechanism. Here, we show that endocytosis and protein abundance of the Arabidopsis brassinosteroid (BR) receptor, BR INSENSITIVE1 (BRI1), are regulated by plant U-box (PUB) E3 ubiquitin ligase PUB12- and PUB13-mediated ubiquitination. BR perception promotes BRI1 ubiquitination and association with PUB12 and PUB13 through phosphorylation at serine 344 residue. Loss of PUB12 and PUB13 results in reduced BRI1 ubiquitination and internalization accompanied with a prolonged BRI1 PM-residence time, indicating that ubiquitination of BRI1 by PUB12 and PUB13 is a key step in BRI1 endocytosis. Our studies provide a molecular link between BRI1 ubiquitination and internalization and reveal a unique mechanism of E3 ligase–substrate association regulated by phosphorylation.

Sequence-Specific Protein Aggregation Generates Defined Protein Knockdowns in Plants

A protein knockdown method based on expression of specific aggregation-prone peptides derived from the targeted proteins. Protein aggregation is determined by short (5–15 amino acids) aggregation-prone regions (APRs) of the polypeptide sequence that self-associate in a specific manner to form β-structured inclusions. Here, we demonstrate that the sequence specificity of APRs can be exploited to selectively knock down proteins with different localization and function in plants. Synthetic aggregation-prone peptides derived from the APRs of either the negative regulators of the brassinosteroid (BR) signaling, the glycogen synthase kinase 3/Arabidopsis SHAGGY-like kinases (GSK3/ASKs), or the starch-degrading enzyme α-glucan water dikinase were designed. Stable expression of the APRs in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) induced aggregation of the target proteins, giving rise to plants displaying constitutive BR responses and increased starch content, respectively. Overall, we show that the sequence specificity of APRs can be harnessed to generate aggregation-associated phenotypes in a targeted manner in different subcellular compartments. This study points toward the potential application of induced targeted aggregation as a useful tool to knock down protein functions in plants and, especially, to generate beneficial traits in crops.