Structure and function of the Arabidopsis ABC transporter ABCB19 in brassinosteroid export.

Brassinosteroids are steroidal phytohormones that regulate plant development and physiology, including adaptation to environmental stresses. Brassinosteroids are synthesized in the cell interior but bind receptors at the cell surface, necessitating a yet to be identified export mechanism. Here, we show that a member of the ATP-binding cassette (ABC) transporter superfamily, ABCB19, functions as a brassinosteroid exporter. We present its structure in both the substrate-unbound and the brassinosteroid-bound states. Bioactive brassinosteroids are potent activators of ABCB19 ATP hydrolysis activity, and transport assays showed that ABCB19 transports brassinosteroids. In Arabidopsis thaliana, ABCB19 and its close homolog, ABCB1, positively regulate brassinosteroid responses. Our results uncover an elusive export mechanism for bioactive brassinosteroids that is tightly coordinated with brassinosteroid signaling.

Adenosine monophosphate deaminase modulates BIN2 activity through hydrogen peroxide-induced oligomerization.

The Arabidopsis thaliana GSK3-like kinase, BRASSINOSTEROID-INSENSITIVE2 (BIN2) is a key negative regulator of brassinosteroid (BR) signaling and a hub for crosstalk with other signaling pathways. However, the mechanisms controlling BIN2 activity are not well understood. Here we performed a forward genetic screen for resistance to the plant-specific GSK3 inhibitor bikinin and discovered that a mutation in the ADENOSINE MONOPHOSPHATE DEAMINASE (AMPD)/EMBRYONIC FACTOR1 (FAC1) gene reduces the sensitivity of Arabidopsis seedlings to both bikinin and BRs. Further analyses revealed that AMPD modulates BIN2 activity by regulating its oligomerization in a hydrogen peroxide (H2O2)-dependent manner. Exogenous H2O2 induced the formation of BIN2 oligomers with a decreased kinase activity and an increased sensitivity to bikinin. By contrast, AMPD activity inhibition reduced the cytosolic reactive oxygen species (ROS) levels and the amount of BIN2 oligomers, correlating with the decreased sensitivity of Arabidopsis plants to bikinin and BRs. Furthermore, we showed that BIN2 phosphorylates AMPD to possibly alter its function. Our results uncover the existence of an H2O2 homeostasis-mediated regulation loop between AMPD and BIN2 that fine-tunes the BIN2 kinase activity to control plant growth and development.

Tripartite hormonal regulation of plasma membrane H+-ATPase activity.

The enzyme activity of the plasma membrane (PM) proton pump, well known as arabidopsis PM H+-ATPase (AHA) in the model plant arabidopsis (Arabidopsis thaliana), is controlled by phosphorylation. Three different classes of phytohormones, brassinosteroids (BRs), abscisic acid (ABA), and auxin regulate plant growth and responses to environmental stimuli, at least in part by modulating the activity of the pump through phosphorylation of the penultimate Thr residue in its carboxyl terminus. Here, we review the current knowledge regarding this tripartite hormonal AHA regulation and highlight mechanisms of activation and deactivation, as well as the significance of hormonal crosstalk. Understanding the complexity of PM H+-ATPase regulation in plants might provide new strategies for sustainable agriculture.

GOLVEN peptide signalling through RGI receptors and MPK6 restricts asymmetric cell division during lateral root initiation.

During lateral root initiation, lateral root founder cells undergo asymmetric cell divisions that generate daughter cells with different sizes and fates, a prerequisite for correct primordium organogenesis. An excess of the GLV6/RGF8 peptide disrupts these initial asymmetric cell divisions, resulting in more symmetric divisions and the failure to achieve lateral root organogenesis. Here, we show that loss-of-function GLV6 and its homologue GLV10 increase asymmetric cell divisions during lateral root initiation, and we identified three members of the RGF1 INSENSITIVE/RGF1 receptor subfamily as likely GLV receptors in this process. Through a suppressor screen, we found that MITOGEN-ACTIVATED PROTEIN KINASE6 is a downstream regulator of the GLV pathway. Our data indicate that GLV6 and GLV10 act as inhibitors of asymmetric cell divisions and signal through RGF1 INSENSITIVE receptors and MITOGEN-ACTIVATED PROTEIN KINASE6 to restrict the number of initial asymmetric cell divisions that take place during lateral root initiation.

Proteolytic Processing of SERK3/BAK1 Regulates Plant Immunity, Development, and Cell Death.

Plants have evolved many receptor-like kinases (RLKs) to sense extrinsic and intrinsic cues. The signaling pathways mediated by multiple Leucine-rich repeat (LRR) RLK (LRR-RLK) receptors require ligand-induced receptor-coreceptor heterodimerization and transphosphorylation with BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1)/SOMATIC EMBRYOGENESIS RECEPTOR KINASES family LRR-RLKs. Here we reveal an additional layer of regulation of BAK1 via a Ca2+-dependent proteolytic cleavage process that is conserved in Arabidopsis (Arabidopsis thaliana), Nicotiana benthamiana, and Saccharomyces cerevisiae The proteolytic cleavage of BAK1 is intrinsically regulated in response to developmental cues and immune stimulation. The surface-exposed Asp (D287) residue of BAK1 is critical for its proteolytic cleavage and plays an essential role in BAK1-regulated plant immunity, growth hormone brassinosteroid-mediated responses, and cell death containment. BAK1D287A mutation impairs BAK1 phosphorylation on its substrate BOTRYTIS-INDUCED KINASE1 (BIK1), and its plasma membrane localization. Intriguingly, it aggravates BAK1 overexpression-triggered cell death independent of BIK1, suggesting that maintaining homeostasis of BAK1 through a proteolytic process is crucial to control plant growth and immunity. Our data reveal that in addition to layered transphosphorylation in the receptor complexes, the proteolytic cleavage is an important regulatory process for the proper functions of the shared coreceptor BAK1 in diverse cellular signaling pathways.

Selective Knockdowns in Maize by Sequence-Specific Protein Aggregation.

Protein aggregation is determined by 5-15 amino acids peptides of the target protein sequence, so-called aggregation-prone regions (APRs) that specifically self-associate to form ß-structured inclusions. The presence of APRs in a target protein can be predicted by a dedicated algorithm, such as TANGO. Synthetic aggregation-prone proteins are designed by expressing specific APRs fused to a fluorescent carrier for stability and visualization. Previously, the stable expression of these proteins in Zea mays (maize) has been demonstrated to induce aggregation of target proteins with specific localization, such as the starch-degrading enzyme α-glucan water dikinase, giving rise to plants displaying knockdown phenotypes. Here, we describe how to design synthetic aggregation-prone proteins to harness the sequence specificity of APRs to generate aggregation-associated phenotypes in a targeted manner and in different subcellular compartments. This method points toward the application of induced targeted aggregation as a useful tool to knock down protein functions in maize and to generate crops with improved traits.

Danger-associated peptide signaling in Arabidopsis requires clathrin.

The Arabidopsis thaliana endogenous elicitor peptides (AtPeps) are released into the apoplast after cellular damage caused by pathogens or wounding to induce innate immunity by direct binding to the membrane-localized leucine-rich repeat receptor kinases, PEP RECEPTOR1 (PEPR1) and PEPR2. Although the PEPR-mediated signaling components and responses have been studied extensively, the contributions of the subcellular localization and dynamics of the active PEPRs remain largely unknown. We used live-cell imaging of the fluorescently labeled and bioactive pep1 to visualize the intracellular behavior of the PEPRs in the Arabidopsis root meristem. We found that AtPep1 decorated the plasma membrane (PM) in a receptor-dependent manner and cointernalized with PEPRs. Trafficking of the AtPep1-PEPR1 complexes to the vacuole required neither the trans-Golgi network/early endosome (TGN/EE)-localized vacuolar H(+)-ATPase activity nor the function of the brefeldin A-sensitive ADP-ribosylation factor-guanine exchange factors (ARF-GEFs). In addition, AtPep1 and different TGN/EE markers colocalized only rarely, implying that the intracellular route of this receptor-ligand pair is largely independent of the TGN/EE. Inducible overexpression of the Arabidopsis clathrin coat disassembly factor, Auxilin2, which inhibits clathrin-mediated endocytosis (CME), impaired the AtPep1-PEPR1 internalization and compromised AtPep1-mediated responses. Our results show that clathrin function at the PM is required to induce plant defense responses, likely through CME of cell surface-located signaling components.

Mitochondrial uncouplers inhibit clathrin-mediated endocytosis largely through cytoplasmic acidification.

ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. Mitochondrial uncouplers dissipate this proton gradient and disrupt numerous cellular processes, including vesicular trafficking, mainly through energy depletion. Here we show that Endosidin9 (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. We show that the known tyrosine kinase inhibitor tyrphostinA23, which is routinely used to block CME, displays similar properties, thus questioning its use as a specific inhibitor of cargo recognition by the AP-2 adaptor complex via tyrosine motif-based endocytosis signals. Furthermore, we show that cytoplasm acidification dramatically affects the dynamics and recruitment of clathrin and associated adaptors, and leads to reduction of phosphatidylinositol 4,5-biphosphate from the plasma membrane.

Sequence-Specific Protein Aggregation Generates Defined Protein Knockdowns in Plants.

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 ß-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.

A novel endosomal sorting complex required for transport (ESCRT) component in Arabidopsis thaliana controls cell expansion and development.

ESCRT proteins mediate membrane remodeling and scission events and are essential for endosomal sorting of plasma membrane proteins for degradation. We have identified a novel, plant-specific ESCRT component called PROS (POSITIVE REGULATOR OF SKD1) in Arabidopsis thaliana. PROS has a strong positive effect on the in vitro ATPase activity of SKD1 (also known as Vacuolar Protein Sorting 4 or VPS4), a critical component required for ESCRT-III disassembly and endosomal vesiculation. PROS interacts with both SKD1 and the SKD1-positive regulator LIP5/VTA1. We have identified a putative MIM domain within PROS that mediate the interaction with the MIT domain of SKD1. Interestingly, whereas MIM domains are commonly found at the C terminus of ESCRT-III subunits, the PROS MIM domain is internal. The heterologous expression of PROS in yeast mutant cells lacking Vta1p partially rescues endosomal sorting defects. PROS is expressed in most tissues and cells types in Arabidopsis thaliana. Silencing of PROS leads to reduced cell expansion and abnormal organ growth.

Emission spectra profiling of fluorescent proteins in living plant cells.

BACKGROUND: Fluorescence imaging at high spectral resolution allows the simultaneous recording of multiple fluorophores without switching optical filters, which is especially useful for time-lapse analysis of living cells. The collected emission spectra can be used to distinguish fluorophores by a computation analysis called linear unmixing. The availability of accurate reference spectra for different fluorophores is crucial for this type of analysis. The reference spectra used by plant cell biologists are in most cases derived from the analysis of fluorescent proteins in solution or produced in animal cells, although these spectra are influenced by both the cellular environment and the components of the optical system. For instance, plant cells contain various autofluorescent compounds, such as cell wall polymers and chlorophyll, that affect the spectral detection of some fluorophores. Therefore, it is important to acquire both reference and experimental spectra under the same biological conditions and through the same imaging systems. RESULTS: Entry clones (pENTR) of fluorescent proteins (FPs) were constructed in order to create C- or N-terminal protein fusions with the MultiSite Gateway recombination technology. The emission spectra for eight FPs, fused C-terminally to the A- or B-type cyclin dependent kinases (CDKA;1 and CDKB1;1) and transiently expressed in epidermal cells of tobacco (Nicotiana benthamiana), were determined by using the Olympus FluoView™ FV1000 Confocal Laser Scanning Microscope. These experimental spectra were then used in unmixing experiments in order to separate the emission of fluorophores with overlapping spectral properties in living plant cells. CONCLUSIONS: Spectral imaging and linear unmixing have a great potential for efficient multicolor detection in living plant cells. The emission spectra for eight of the most commonly used FPs were obtained in epidermal cells of tobacco leaves and used in unmixing experiments. The generated set of FP Gateway entry vectors represents a valuable resource for plant cell biologists.

Ectopic expression of Kip-related proteins restrains root-knot nematode-feeding site expansion.

The development of nematode feeding sites induced by root-knot nematodes involves the synchronized activation of cell cycle processes such as acytokinetic mitoses and DNA amplification. A number of key cell cycle genes are reported to be critical for nematode feeding site development. However, it remains unknown whether plant cyclin-dependent kinase (CDK) inhibitors such as the Arabidopsis interactor/inhibitor of CDK (ICK)/Kip-related protein (KRP) family are involved in nematode feeding site development. This study demonstrates the involvement of Arabidopsis ICK2/KRP2 and ICK1/KRP1 in the control of mitosis to endoreduplication in galls induced by the root-knot nematode Meloidogyne incognita. Using ICK/KRP promoter-GUS fusions and mRNA in situ hybridizations, we showed that ICK2/KRP2, ICK3/KRP5 and ICK4/KRP6 are expressed in galls after nematode infection. Loss-of-function mutants have minor effects on gall development and nematode reproduction. Conversely, overexpression of both ICK1/KRP1 and ICK2/KRP2 impaired mitosis in giant cells and blocked neighboring cell proliferation, resulting in a drastic reduction of gall size. Studying the dynamics of protein expression demonstrated that protein levels of ICK2/KRP2 are tightly regulated during giant cell development and reliant on the presence of the nematode. This work demonstrates that impeding cell cycle progression by means of ICK1/KRP1 and ICK2/KRP2 overexpression severely restricts gall development, leading to a marked limitation of root-knot nematode development and reduced numbers of offspring.

Clusters of bioactive compounds target dynamic endomembrane networks in vivo.

Endomembrane trafficking relies on the coordination of a highly complex, dynamic network of intracellular vesicles. Understanding the network will require a dissection of cargo and vesicle dynamics at the cellular level in vivo. This is also a key to establishing a link between vesicular networks and their functional roles in development. We used a high-content intracellular screen to discover small molecules targeting endomembrane trafficking in vivo in a complex eukaryote, Arabidopsis thaliana. Tens of thousands of molecules were prescreened and a selected subset was interrogated against a panel of plasma membrane (PM) and other endomembrane compartment markers to identify molecules that altered vesicle trafficking. The extensive image dataset was transformed by a flexible algorithm into a marker-by-phenotype-by-treatment time matrix and revealed groups of molecules that induced similar subcellular fingerprints (clusters). This matrix provides a platform for a systems view of trafficking. Molecules from distinct clusters presented avenues and enabled an entry point to dissect recycling at the PM, vacuolar sorting, and cell-plate maturation. Bioactivity in human cells indicated the value of the approach to identifying small molecules that are active in diverse organisms for biology and drug discovery.

A high-throughput bimolecular fluorescence complementation protein-protein interaction screen identifies functional Arabidopsis CDKA/B-CYCD4/5 complexes.

The eukaryotic cell cycle is a process controlled by protein assemblies, of which the key subunits are serine-threonine cyclin-dependent kinases (CDKs). Timely association and dissociation of these assemblies ensure that the cell division program is executed correctly. The challenge to unravel the rules of the plant cell cycle results from the multiplicity of the process-regulating genes that emerged through genome duplications during the evolution of flowering plants. Despite the increasing knowledge on the plant cell cycle control, little is known about the composition of the different CDK-Cyclin complexes and their spatio-temporal occurrence. The binary interactions of the previously annotated 58 Arabidopsis thaliana core cell cycle proteins were tested in two high-throughput protein-protein interaction (PPI) assays: the bimolecular fluorescence complementation (BiFC) and the yeast two-hybrid. The resulting PPI network was integrated with available cycle phase-dependent gene expression data and subcellular localization information, revealing distinct cell cycle clusters acting at different cell division stages. Additionally, the BiFC assay revealed that three D-type cyclins, CYCD4;1, CYCD4;2 and CYCD5;1, form active kinase complexes with CDKA;1 and CDKB1;1 in vivo because they induce cell divisions in differentiated tobacco (Nicotiana benthamiana) epidermal cells. We demonstrate that these complexes promote cell proliferation in Arabidopsis and we discuss their putative mode of action in plant development.

Nuclear-localized subtype of end-binding 1 protein regulates spindle organization in Arabidopsis.

End-binding 1 (EB1) proteins are evolutionarily conserved plus-end-tracking proteins that localize to growing microtubule plus ends where they regulate microtubule dynamics and interactions with intracellular targets. Animal EB1 proteins have acidic C-terminal tails that might induce an autoinhibitory conformation. Although EB1 proteins with the same structural features occur in plants (EB1a and EB1b in Arabidopsis thaliana), a variant form (EB1c) is present that lacks the characteristic tail. We show that in Arabidopsis the tail region of EB1b, but not of EB1c, inhibits microtubule assembly in vitro. EB1a and EB1b form heterodimers with each other, but not with EB1c. Furthermore, the EB1 genes are expressed in various cell types of Arabidopsis, but the expression of EB1c is particularly strong in the meristematic cells where it is targeted to the nucleus by a nuclear localization signal in the C-terminal tail. Reduced expression of EB1c compromised the alignment of spindle and phragmoplast microtubules and caused frequent lagging of separating chromosomes at anaphase. Roots of the eb1c mutant were hypersensitive to a microtubule-disrupting drug and complete rescue of the mutant phenotype required the tail region of EB1c. These results suggest that a plant-specific EB1 subtype has evolved to function preferentially on the spindle microtubules by accumulating in the prophase nucleus.

Systematic localization of the Arabidopsis core cell cycle proteins reveals novel cell division complexes.

Cell division depends on the correct localization of the cyclin-dependent kinases that are regulated by phosphorylation, cyclin proteolysis, and protein-protein interactions. Although immunological assays can define cell cycle protein abundance and localization, they are not suitable for detecting the dynamic rearrangements of molecular components during cell division. Here, we applied an in vivo approach to trace the subcellular localization of 60 Arabidopsis (Arabidopsis thaliana) core cell cycle proteins fused to green fluorescent proteins during cell division in tobacco (Nicotiana tabacum) and Arabidopsis. Several cell cycle proteins showed a dynamic association with mitotic structures, such as condensed chromosomes and the preprophase band in both species, suggesting a strong conservation of targeting mechanisms. Furthermore, colocalized proteins were shown to bind in vivo, strengthening their localization-function connection. Thus, we identified unknown spatiotemporal territories where functional cell cycle protein interactions are most likely to occur.

CDKB1;1 forms a functional complex with CYCA2;3 to suppress endocycle onset.

The mitosis-to-endocycle transition requires the controlled inactivation of M phase-associated cyclin-dependent kinase (CDK) activity. Previously, the B-type CDKB1;1 was identified as an important negative regulator of endocycle onset. Here, we demonstrate that CDKB1;1 copurifies and associates with the A2-type cyclin CYCA2;3. Coexpression of CYCA2;3 with CDKB1;1 triggered ectopic cell divisions and inhibited endoreduplication. Moreover, the enhanced endoreduplication phenotype observed after overexpression of a dominant-negative allele of CDKB1;1 could be partially complemented by CYCA2;3 co-overexpression, illustrating that both subunits unite in vivo to form a functional complex. CYCA2;3 protein stability was found to be controlled by CCS52A1, an activator of the anaphase-promoting complex. We conclude that CCS52A1 participates in endocycle onset by down-regulating CDKB1;1 activity through the destruction of CYCA2;3.

The Arabidopsis SERK1 protein interacts with the AAA-ATPase AtCDC48, the 14-3-3 protein GF14lambda and the PP2C phosphatase KAPP.

Leucine-rich repeat (LRR)-containing transmembrane receptor-like kinases (RLKs) are important components of plant signal transduction. The Arabidopsis thaliana somatic embryogenesis receptor-like kinase 1 (AtSERK1) is an LRR-RLK proposed to participate in a signal transduction cascade involved in embryo development. By yeast two-hybrid screening we identified AtCDC48, a homologue of the mammalian AAA-ATPase p97 and GF14lambda, a member of the Arabidopsis family of 14-3-3 proteins as AtSERK1 interactors. In vitro, the AtSERK1 kinase domain is able to transphosphorylate and bind both AtCDC48 and GF14lambda. In yeast, AtCDC48 interacts with GF14lambda and with the PP2C phosphatase KAPP. In plant protoplasts AtSERK1 interacts with GF14lambda.