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Aluminum (Al) toxicity and inorganic phosphate (Pi) limitation are widespread chronic abiotic and mutually enhancing stresses that profoundly affect crop yield. Both stresses cause a strong inhibition of root growth, resulting from a progressive exhaustion of the stem cell niche. Here, we report on a casein kinase 2 (CK2) inhibitor identified by its capability to maintain a functional root stem cell niche under Al toxic conditions. CK2 operates through phosphorylation of the cell cycle checkpoint activator SUPPRESSOR OF GAMMA RADIATION1 (SOG1), priming its activity under DNA-damaging conditions. In addition to yielding Al tolerance, CK2 and SOG1 inactivation prevents meristem exhaustion under Pi starvation, revealing the existence of a low Pi-induced cell cycle checkpoint that depends on the DNA damage activator ATAXIA-TELANGIECTASIA MUTATED. Overall, our data reveal an important physiological role for the plant DNA damage response pathway under agriculturally limiting growth conditions, opening new avenues to cope with Pi limitation. ONE-SENTENCE SUMMARY Casein kinase 2 and DNA damage response regulators play a pivotal role in the control of Arabidopsis root growth in response to Al toxicity and phosphate limitation.

The proteome and phosphoproteome of CEP5 overexpressing Arabidopsis seedlings have been determined. This revealed that CEP5 impacts abiotic stress-related processes. Subsequent genetic, physiological, biochemical, and pharmacological results demonstrated that CEP5-mediated signaling is relevant for osmotic and drought stress tolerance in Arabidopsis. Furthermore, CEP5 specifically counteracts auxin effects by stabilizing AUX/IAA transcriptional repressors., Graphical Abstract Highlights • Quantitative Arabidopsis (phospho)proteomes of C-TERMINALLY ENCODED PEPTIDE 5 (CEP5). • CEP5 impacts abiotic stress-related processes and counteracts auxin effects. • CEP5 signaling stabilizes AUX/IAA transcriptional repressors. • Novel peptide-dependent control mechanism that tunes auxin signaling., Peptides derived from non-functional precursors play important roles in various developmental processes, but also in (a)biotic stress signaling. Our (phospho)proteome-wide analyses of C-TERMINALLY ENCODED PEPTIDE 5 (CEP5)-mediated changes revealed an impact on abiotic stress-related processes. Drought has a dramatic impact on plant growth, development and reproduction, and the plant hormone auxin plays a role in drought responses. Our genetic, physiological, biochemical, and pharmacological results demonstrated that CEP5-mediated signaling is relevant for osmotic and drought stress tolerance in Arabidopsis, and that CEP5 specifically counteracts auxin effects. Specifically, we found that CEP5 signaling stabilizes AUX/IAA transcriptional repressors, suggesting the existence of a novel peptide-dependent control mechanism that tunes auxin signaling. These observations align with the recently described role of AUX/IAAs in stress tolerance and provide a novel role for CEP5 in osmotic and drought stress tolerance.

Clathrin-mediated endocytosis (CME) is a highly conserved and essential cellular process in eukaryotic cells, but its dynamic and vital nature makes it challenging to study using classical genetics tools. In contrast, although small molecules can acutely and reversibly perturb CME, the few chemical CME inhibitors that have been applied to plants are either ineffective or show undesirable side effects. Here, we identify the previously described endosidin9 (ES9) as an inhibitor of clathrin heavy chain (CHC) function in both Arabidopsis and human cells through affinity-based target isolation, in vitro binding studies and X-ray crystallography. Moreover, we present a chemically improved ES9 analog, ES9-17, which lacks the undesirable side effects of ES9 while retaining the ability to target CHC. ES9 and ES9-17 have expanded the chemical toolbox used to probe CHC function, and present chemical scaffolds for further design of more specific and potent CHC inhibitors across different systems.

Many signal perception mechanisms are connected to Ca2+-based second messenger signaling to modulate specific cellular responses. The well-characterized plant hormone auxin elicits a very rapid Ca2+ signal. However, the cellular targets of auxin-induced Ca2+ are largely unknown. Here, we screened a biologically annotated chemical library for inhibitors of auxin-induced Ca2+ entry in plant cell suspensions to better understand the molecular mechanism of auxin-induced Ca2+ and to explore the physiological relevance of Ca2+ in auxin signal transduction. Using this approach, we defined a set of diverse, small molecules that interfere with auxin-induced Ca2+ entry. Based on annotated biological activities of the hit molecules, we found that auxin-induced Ca2+ signaling is, among others, highly sensitive to disruption of membrane proton gradients and the mammalian Ca2+ channel inhibitor bepridil. Whereas protonophores nonselectively inhibited auxin-induced and osmotic stress-induced Ca2+ signals, bepridil specifically inhibited auxin-induced Ca2+ We found evidence that bepridil severely alters vacuolar morphology and antagonized auxin-induced vacuolar remodeling. Further exploration of this plant-tailored collection of inhibitors will lead to a better understanding of auxin-induced Ca2+ entry and its relevance for auxin responses.

Plant secondary-thickened cell walls are characterized by the presence of lignin, a recalcitrant and hydrophobic polymer that provides mechanical strength and ensures long-distance water transport. Exactly the recalcitrance and hydrophobicity of lignin put a burden on the industrial processing efficiency of lignocellulosic biomass. Both forward and reverse genetic strategies have been used intensively to unravel the molecular mechanism of lignin deposition. As an alternative strategy, we introduce here a forward chemical genetic approach to find candidate inhibitors of lignification. A high-throughput assay to assess lignification in Arabidopsis (Arabidopsis thaliana) seedlings was developed and used to screen a 10-k library of structurally diverse, synthetic molecules. Of the 73 compounds that reduced lignin deposition, 39 that had a major impact were retained and classified into five clusters based on the shift they induced in the phenolic profile of Arabidopsis seedlings. One representative compound of each cluster was selected for further lignin-specific assays, leading to the identification of an aromatic compound that is processed in the plant into two fragments, both having inhibitory activity against lignification. One fragment, p-iodobenzoic acid, was further characterized as a new inhibitor of CINNAMATE 4-HYDROXYLASE, a key enzyme of the phenylpropanoid pathway synthesizing the building blocks of the lignin polymer. As such, we provide proof of concept of this chemical biology approach to screen for inhibitors of lignification and present a broad array of putative inhibitors of lignin deposition for further characterization.

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., Plant cells maintain strict proton gradients over different membranes. Here, Dejonghe et al. show that several protonophores, including the known tyrosine kinase inhibitor TyrphostinA23, inhibit clathrin-mediated endocytosis by disturbing these gradients and causing cytoplasmic acidification.

International audience; Small GTP-binding proteins from the ADP-ribosylation factor (ARF) family are important regulators of vesicle formation and cellular trafficking in all eukaryotes. ARF activation is accomplished by a protein family of guanine nucleotide exchange factors (GEFs) that contain a conserved catalytic Sec7 domain. Here, we identified and characterized Secdin, a small-molecule inhibitor of Arabidopsis thaliana ARF-GEFs. Secdin application caused aberrant retention of plasma membrane (PM) proteins in late endosomal compartments, enhanced vacuolar degradation, impaired protein recycling, and delayed secretion and endocytosis. Combined treatments with Secdin and the known ARF-GEF inhibitor Brefeldin A (BFA) prevented the BFA-induced PM stabilization of the ARF-GEF GNOM, impaired its translocation from the Golgi to the trans-Golgi network/early endosomes, and led to the formation of hybrid endomembrane compartments reminiscent of those in ARF-GEF-deficient mutants. Drug affinity-responsive target stability assays revealed that Secdin, unlike BFA, targeted all examined Arabidopsis ARF-GEFs, but that the interaction was probably not mediated by the Sec7 domain because Secdin did not interfere with the Sec7 domain-mediated ARF activation. These results show that Secdin and BFA affect their protein targets through distinct mechanisms, in turn showing the usefulness of Secdin in studies in which ARF-GEF-dependent endomembrane transport cannot be manipulated with BFA.

The high metabolic flux through photorespiration constitutes a significant part of the carbon cycle. Although the major enzymatic steps of the photorespiratory pathway are well characterized, little information is available on the functional significance of photorespiration beyond carbon recycling. Particularly important in this respect is the peroxisomal catalase activity which removes photorespiratory H2O2 generated during the oxidation of glycolate to glyoxylate, thus maintaining the cellular redox homeostasis governing the perception, integration and execution of stress responses. By performing a chemical screen, we identified 34 small molecules that alleviate the negative effects of photorespiration in Arabidopsis thaliana mutants lacking photorespiratory catalase (cat2). The chlorophyll fluorescence parameter photosystem II maximum efficiency (Fv′/Fm′) was used as a high-throughput readout. The most potent chemical that could rescue the photorespiratory phenotype of cat2 is a pro-auxin that contains a synthetic auxin-like substructure belonging to the phenoxy herbicide family, which can be released in planta. The naturally occurring indole-3-acetic acid (IAA) and other chemically distinct synthetic auxins also inhibited the photorespiratory-dependent cell death in cat2 mutants, implying a role for auxin signalling in stress tolerance.

The application of small molecules has played a crucial role in identifying novel components involved in plant signalling. Compared to classic genetic approaches, small molecule screens offer notable advantages in dissecting plant biological processes, such as technical simplicity, low start-up costs, and most importantly, bypassing the problems of lethality and redundancy. To identify small molecules that target a biological process or protein of interest, robust and well-reasoned high-throughput screening approaches are essential. In this review, we present a series of principles and valuable approaches in small molecule screening in the plant model system Arabidopsis thaliana. We also provide an overview of small molecules that led to breakthroughs in uncovering phytohormone signalling pathways, endomembrane signalling cascades, novel growth regulators, and plant defence mechanisms. Meanwhile, the strategies to deciphering the mechanisms of these small molecules on Arabidopsis are highlighted. Moreover, the opportunities and challenges of small molecule applications in translational biology are discussed.

Because the plant cell wall provides the first line of defense against biotic and abiotic assaults, its functional integrity needs to be maintained under stress conditions. Through a phenotype-based compound screening approach, we identified a novel cellulose synthase inhibitor, designated C17. C17 administration depletes cellulose synthase complexes from the plasma membrane in Arabidopsis thaliana, resulting in anisotropic cell elongation and a weak cell wall. Surprisingly, in addition to mutations in CELLULOSE SYNTHASE1 (CESA1) and CESA3, a forward genetic screen identified two independent defective genes encoding pentatricopeptide repeat (PPR)-like proteins (CELL WALL MAINTAINER1 [CWM1] and CWM2) as conferring tolerance to C17. Functional analysis revealed that mutations in these PPR proteins resulted in defective cytochrome c maturation and activation of mitochondrial retrograde signaling, as evidenced by the induction of an alternative oxidase. These mitochondrial perturbations increased tolerance to cell wall damage induced by cellulose deficiency. Likewise, administration of antimycin A, an inhibitor of mitochondrial complex III, resulted in tolerance toward C17. The C17 tolerance of cwm2 was partially lost upon depletion of the mitochondrial retrograde regulator ANAC017, demonstrating that ANAC017 links mitochondrial dysfunction with the cell wall. In view of mitochondria being a major target of a variety of stresses, our data indicate that plant cells might modulate mitochondrial activity to maintain a functional cell wall when subjected to stresses.

Cell death establishes a site for development As plant roots grow through the soil, lateral roots emerge to reach more resources. Xuan et al. now show that programmed cell death sets the course for lateral root development. Cells in a specialized region of the root cap periodically die off as a group, defining a location at which a lateral root will later develop. Science , this issue p. 384

Highlight We identified C-TERMINALLY ENCODED PEPTIDE 5 (CEP5) as a novel, auxin-repressed and phloem pole-expressed signal assisting in the formation of lateral roots., Roots explore the soil for water and nutrients through the continuous production of lateral roots. Lateral roots are formed at regular distances in a steadily elongating organ, but how future sites for lateral root formation become established is not yet understood. Here, we identified C-TERMINALLY ENCODED PEPTIDE 5 (CEP5) as a novel, auxin-repressed and phloem pole-expressed signal assisting in the formation of lateral roots. In addition, based on genetic and expression data, we found evidence for the involvement of its proposed receptor, XYLEM INTERMIXED WITH PHLOEM 1 (XIP1)/CEP RECEPTOR 1 (CEPR1), during the process of lateral root initiation. In conclusion, we report here on the existence of a peptide ligand−receptor kinase interaction that impacts lateral root initiation. Our results represent an important step towards the understanding of the cellular communication implicated in the early phases of lateral root formation.

International audience; Global climate change and a growing population require tackling the reduction in arable land and improving biomass production and seed yield per area under varying conditions. One of these conditions is suboptimal water availability. Here, we review some of the classical approaches to dealing with plant response to drought stress and we evaluate how research on RECEPTOR-LIKE KINASES (RLKs) can contribute to improving plant performance under drought stress. RLKs are considered as key regulators of plant architecture and growth behavior, but they also function in defense and stress responses. The available literature and analyses of available transcript profiling data indeed suggest that RLKs can play an important role in optimizing plant responses to drought stress. In addition, RLK pathways are ideal targets for nontransgenic approaches, such as synthetic molecules, providing a novel strategy to manipulate their activity and supporting translational studies from model species, such as Arabidopsis thaliana, to economically useful crops.

Research into the plant hormone auxin has always been tightly linked with the use of small molecules. In fact, most of the known players in auxin signaling and transport in the model plant Arabidopsis thaliana were identified by screening for resistance to auxin analogues. The use of high-throughput screening technologies has since yielded many novel molecules, opening the way for the identification of new target proteins to further elucidate known pathways. Here, we give an overview of well-established and novel molecules used in auxin research and highlight the current status and future perspectives of chemical biology approaches to auxin biology.

In plants, the developmental mechanisms that regulate the positioning of lateral organs along the primary root are currently unknown. We present evidence on how lateral root initiation is controlled in a spatiotemporal manner in the model plant Arabidopsis thaliana. First, lateral roots are spaced along the main axis in a regular left-right alternating pattern that correlates with gravity-induced waving and depends on AUX1, an auxin influx carrier essential for gravitropic response. Second, we found evidence that the priming of pericycle cells for lateral root initiation might take place in the basal meristem, correlating with elevated auxin sensitivity in this part of the root. This local auxin responsiveness oscillates with peaks of expression at regular intervals of 15 hours. Each peak in the auxin-reporter maximum correlates with the formation of a consecutive lateral root. Third, auxin signaling in the basal meristem triggers pericycle cells for lateral root initiation prior to the action of INDOLE-3-ACETIC ACID14(SOLITARY ROOT).

Mutations in the gene encoding the gamma2 subunit of the gamma-aminobutyric acid type A receptor (GABRG2) have been reported to cause childhood absence epilepsy (CAE), febrile seizures (FS), and generalized epilepsy with FS plus (GEFS+). The authors analyzed GABRG2 in 47 unrelated patients with CAE, FS, and GEFS+ and identified a novel mutation that cosegregated with FS. Electrophysiologic studies demonstrated altered current desensitization and reduced benzodiazepine enhancement in mutant receptors.

Febrile seizures (FS) represent the most common seizure disorder in childhood and contribution of a genetic predisposition has been clearly proven. In some families FS is associated with a wide variety of afebrile seizures. Generalized epilepsy with febrile seizures plus (GEFS+) is a familial epilepsy syndrome with a spectrum of phenotypes including FS, atypical febrile seizures (FS+) and afebrile generalized and partial seizures. Mutations in the genes SCN1B, SCN1A and GABRG2 were identified in GEFS+ families. GEFS+ is genetically heterogeneous and mutations in these three genes were detected in only a minority of the families. We performed a 10 cM density genome-wide scan in a multigenerational family with febrile seizures and epilepsy and obtained a maximal multipoint LOD score of 3.12 with markers on chromosome 5q14.3-q23.1. Fine mapping and segregation analysis defined a genetic interval of approximately 33 cM between D5S2103 and D5S1975. This candidate region overlapped with a previously reported locus for febrile seizures (FEB4) in the Japanese population, in which MASS1 was proposed as disease gene. Mutation analysis of the exons and exon-intron boundaries of MASS1 in our family did not reveal a disease causing mutation. Our linkage data confirm for the first time that a locus on chromosome 5q14-q23 plays a role in idiopathic epilepsies. However, our mutation data is negative and do not support a role for MASS1 suggesting that another gene within or near the FEB4 locus might exist.

Severe myoclonic epilepsy of infancy (SMEI or Dravet syndrome) is a rare disorder occurring in young children often without a family history of a similar disorder. The earliest disease manifestations are usually fever-associated seizures. Later in life, patients display different types of afebrile seizures including myoclonic seizures. Arrest of psychomotor development occurs in the second year of life and most patients become ataxic. Patients are resistant to antiepileptic drug therapy. Recently, we described de novo mutations of the neuronal sodium channel alpha-subunit gene SCN1A in seven isolated SMEI patients. To investigate the contribution of SCN1A mutations to the etiology of SMEI, we examined nine additional SMEI patients. We observed eight coding and one noncoding mutation. In contrast to our previous study, most mutations are missense mutations clustering in the S4-S6 region of SCN1A. These findings demonstrate that de novo mutations in SCN1A are a major cause of isolated SMEI.

Small molecules which act as hormone agonists or antagonists represent useful tools in fundamental research and are widely applied in agriculture to control hormone effects. High-throughput screening of large chemical compound libraries has yielded new findings in plant biology, with possible future applications in agriculture and horticulture. To further understand ethylene biosynthesis/signaling and its crosstalk with other hormones, we screened a 12,000 compound chemical library based on an ethylene-related bioassay of dark-grown Arabidopsis thaliana (L.) Heynh. seedlings. From the initial screening, 1313 (∼11%) biologically active small molecules altering the phenotype triggered by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), were identified. Selection and sorting in classes were based on the angle of curvature of the apical hook, the length and width of the hypocotyl and the root. A MySQL-database was constructed (https://chaos.ugent.be/WE15/) including basic chemical information on the compounds, images illustrating the phenotypes, phenotype descriptions and classification. The research perspectives for different classes of hit compounds will be evaluated, and some general screening tips for customized high-throughput screening and pitfalls will be discussed.

Mitochondria are crucial players in the signaling and metabolic homeostasis of the plant cell. The molecular components that orchestrate the underlying processes, however, are largely unknown. Using a chemical biology approach, we exploited the responsiveness of Arabidopsis UDP-glucosyltransferase-encoding UGT74E2 towards mitochondrial perturbation in order to look for novel mechanisms regulating mitochondria-to-nucleus communication. The most potent inducers of UGT74E2 shared a (2-furyl)acrylate (FAA) substructure that negatively affected mitochondrial function and was identified before as an auxin transcriptional inhibitor. Based on these premises, we demonstrated that perturbed mitochondria negatively affect the auxin signaling machinery. Moreover, chemical perturbation of polar auxin transport and auxin biosynthesis was sufficient to induce mitochondrial retrograde markers and their transcript abundance was constitutively elevated in the absence of the auxin transcriptional activators ARF7 and ARF19.

High-throughput small molecule screenings in model plants are of great value to identify compounds that interfere with plant developmental processes. In academic research, the plant Arabidopsis thaliana is the most commonly used model organism for this purpose. However, compared to plant cellular systems, A. thaliana plants are less amenable to develop high-throughput screening assays. In this chapter, we describe a screening procedure that is compatible with liquid handling systems and increases the throughput of compound screenings in A. thaliana seedlings.

The phytohormone auxin is a key developmental signal in plants. So far, only auxin perception has been described to trigger the release of transcription factors termed Auxin Response Factors (ARFs) from their auxin/indole-3-acetic acid (AUX/IAA) repressor proteins. Here, we show that phosphorylation of ARF7 and ARF19 by BRASSINOSTEROID-insensitive2 (BIN2) can also potentiate auxin signalling output during lateral root organogenesis. BIN2-mediated phosphorylation of ARF7 and ARF19 suppresses their interaction with AUX/IAAs, and subsequently enhances the transcriptional activity to their target genes lateral organ boundaries-domain16 (LBD16) and LBD29. In this context, BIN2 is under the control of the Tracheary element differentiation inhibitory factor (TDIF)-TDIF receptor (TDR) module. TDIF-initiated TDR signalling directly acts on BIN2-mediated ARF phosphorylation, leading to the regulation of auxin signalling during lateral root development. In summary, this study delineates a TDIF-TDR-BIN2 signalling cascade that controls regulation of ARF and AUX/IAA interaction independent of auxin perception during lateral root development.

Development of the root system is essential for proper plant growth and development. Extension of the root system is achieved by the continuous establishment of new meristems in existing parental root tissues, which leads to the development of lateral roots. This process of lateral root organogenesis consists of different developmental stages, which are all controlled by the plant hormone auxin. In this chapter, we describe a screening method in Arabidopsis thaliana to identify small synthetic molecules that interfere with the process of lateral root development during specific developmental stages.

The acquisition of water and nutrients by plant roots is a fundamental aspect of agriculture and strongly depends on root architecture. Root branching and expansion of the root system is achieved through the development of lateral roots and is to a large extent controlled by the plant hormone auxin. However, the pleiotropic effects of auxin or auxin-like molecules on root systems complicate the study of lateral root development. Here we describe a small-molecule screen in Arabidopsis thaliana that identified naxillin as what is to our knowledge the first non-auxin-like molecule that promotes root branching. By using naxillin as a chemical tool, we identified a new function for root cap-specific conversion of the auxin precursor indole-3-butyric acid into the active auxin indole-3-acetic acid and uncovered the involvement of the root cap in root branching. Delivery of an auxin precursor in peripheral tissues such as the root cap might represent an important mechanism shaping root architecture.

The growth-promoting hormones, the brassinosteroids (BRs), are perceived at the plant cell surface by receptor kinases that transduce the signal to the nucleus by an intracellular cascade of phosphorylation-mediated protein-protein interactions. BR signaling is also regulated by the plant endocytic machinery because the increased endosomal localization of the BR receptor enhances the BR responses. Chemical genetics is a powerful approach to identify new components in redundant signaling networks and to characterize highly dynamic processes, such as endocytosis. Here, we describe a screen in Arabidopsis thaliana seedlings for small molecules that affect hypocotyl elongation under continuous light conditions, indicative for an effect on BR responses. The compounds identified in this screen were used to dissect endomembrane trafficking of the BR receptor, BR INSENSITIVE1, a process that is essential for BR signal transduction.

Summary Background Lateral roots are formed at regular intervals along the main root by recurrent specification of founder cells. To date, the mechanism by which branching of the root system is controlled and founder cells become specified remains unknown. Results Our study reports the identification of the auxin regulatory components and their target gene, GATA23 , which control lateral root founder cell specification. Initially, a meta-analysis of lateral root-related transcriptomic data identified the GATA23 transcription factor. GATA23 is expressed specifically in xylem pole pericycle cells before the first asymmetric division and is correlated with oscillating auxin signaling maxima in the basal meristem. Also, functional studies revealed that GATA23 controls lateral root founder cell identity. Finally, we show that an Aux/IAA28-dependent auxin signaling mechanism in the basal meristem controls GATA23 expression. Conclusions We have identified the first molecular components that control lateral root founder cell identity in the Arabidopsis root. These include an IAA28-dependent auxin signaling module in the basal meristem region that regulates GATA23 expression and thereby lateral root founder cell specification and root branching patterns.

Severe myoclonic epilepsy of infancy (SMEI) or Dravet syndrome is a rare epilepsy syndrome. In 30 to 70% of SMEI patients, truncating and missense mutations in the neuronal voltage-gated sodium-channel α-subunit gene (SCN1A) have been identified. The majority of patients have truncating mutations that are predicted to be loss-of-function alleles. Because mutation detection studies use PCR-based sequencing or conformation sensitive gel electrophoresis (CSGE), microdeletions, which are also predicted to be loss-of-function alleles, can easily escape detection. We selected 11 SMEI patients with or without additional features who had no SCN1A mutation detectable with sequencing analysis. In addition, none of the patients was heterozygous for any of the SNPs in SCN1A, indicating that they were either homozygous for all SNPs or hemizygous due to a microdeletion of the gene. We subsequently analyzed these patients for the presence of microdeletions in SCN1A using a quantitative PCR method named multiplex amplicon quantification (MAQ), and observed three patients missing one copy of the SCN1A gene. All three microdeletions were confirmed by fluorescence in situ hybridization (FISH). These findings demonstrate that a substantial percentage of SCN1A-mutation-negative SMEI patients with or without additional features carry a chromosomal microdeletion comprising the SCN1A gene and that haploinsufficiency of the SCN1A gene is a cause of SMEI. Hum Mutat 27(9), 914–920, 2006. © 2006 Wiley-Liss, Inc.

Epilepsy is a paroxysmal disorder with a cumulative incidence of about 3%. About 13% of patients with epilepsy have a history of febrile seizures (FS). Generalized epilepsy with FS plus (GEFS+) is a familial epilepsy syndrome in which patients can have classic FS, FS that persist beyond the age of 5 years (i.e., FS+), and/or epilepsy. Both genetic and environmental factors have been shown to contribute to the pathogenesis of FS and GEFS+. During the past 10 years, molecular genetic studies have contributed a great deal to the identification of genetic factors involved in FS and GEFS+. In this study we aimed to provide a comprehensive review of currently known genes for FS and GEFS+, and the methods and approaches used to identify them. We also discuss the knowledge we currently have and hypotheses regarding the effect of the mutations on their respective protein functions.

Generalised epilepsy with febrile seizures plus (GEFS+) is a clinically and genetically heterogeneous epilepsy syndrome. Using positional cloning strategies, mutations in SCN1B, SCN1A, and GABRG2 have been identified as genetic causes of GEFS+. In the present study, we describe a large four generation family with GEFS+ in which we performed a 10 cM density genome-wide scan. We obtained conclusive evidence for a novel GEFS+ locus on chromosome 2p24 with a maximum two point logarithm of the odds (LOD) score of 4.22 for marker D2S305 at zero recombination. Fine mapping and haplotype segregation analysis in this family delineated a candidate region of 3.24 cM, corresponding to a physical distance of 4.2 Mb. Linkage to 2p24 was confirmed (p = 0.007) in a collection of 50 nuclear and multiplex families with febrile seizures and epilepsy. Transmission disequilibrium testing and association studies provided further evidence (p

Idiopathic epilepsies have a genetic basis and are characterised by the absence of an overt underlying neurological abnormality. Most idiopathic epilepsies are complex diseases with considerable clinical and genetic heterogeneity and an unclear inheritance pattern because of genetic and environmental factors. Families in which the disease segregates as an autosomal dominant trait with reduced disease penetrance have been identified occasionally. In some of these families, a single gene defect was identified as the cause of epilepsy. To date, mutations in 13 genes have been identified for distinct epilepsy types. Most genes encode subunits of ion channels.1,2 In addition, the gene remains to be identified for 21 mapped loci for epilepsy, which highlights the genetic heterogeneity of the idiopathic epilepsy syndromes.3,4 Familial temporal lobe epilepsy (MIM 608096) was first described by Berkovic et al . and was recognised as a distinct epileptic syndrome by the International League Against Epilepsy.5 It is defined by familial occurrence of simple partial seizures, complex partial seizures, and secondarily generalised seizures of temporal lobe origin.6 Two genetically distinct autosomal dominant familial temporal lobe epilepsy syndromes have been reported. Autosomal dominant lateral temporal lobe epilepsy (MIM 600512), or autosomal dominant partial epilepsy with auditory features, was described first by Ottman et al . ,7 and recently, mutations in the leucine rich glioma inactivated 1 ( LGI1 ) gene on chromosome 10q24 were identified.8,9 Auras that present as auditory and visual hallucinations are a clinical hallmark of this syndrome. The other variant of familial temporal lobe epilepsy is characterised clinically by onset in teenage years or early adulthood, absence of antecedent factors, low frequency of deja vu, and a usually good prognosis. This variant, which still can be heterogeneous genetically, is not mapped yet. In a large family with febrile seizures …

Generalized epilepsy with febrile seizures plus (GEFS+) is a clinically and genetically heterogeneous syndrome with childhood onset, characterized by febrile seizures (FS) and a variety of afebrile epileptic seizure types. The authors performed a mutational analysis of SCN1B on 74 unrelated probands with GEFS+, FS, or FS plus (FS+). In a family with FS+ and early-onset absence epilepsy, a mutation was identified that predicts a deletion of five amino acids in the extracellular immunoglobulin-like domain of SCN1B and potential loss of function. SCN1B mutations are associated with GEFS+ and may have a role in the elicitation of absence seizures.

SummaryDuring the exploration of the soil by plant roots, uptake of water and nutrients can be greatly fostered by a regular spacing of lateral roots (LRs). In the Arabidopsis root, a regular branching pattern depends on oscillatory gene activity to create prebranch sites, patches of cells competent to form LRs. Thus far, the molecular components regulating the oscillations still remain unclear. Here, we show that a local auxin source in the root cap, derived from the auxin precursor indole-3-butyric acid (IBA), modulates the oscillation amplitude, which in turn determines whether a prebranch site is created or not. Moreover, transcriptome profiling identified novel and IBA-regulated components of root patterning, such as the MEMBRANE-ASSOCIATED KINASE REGULATOR4 (MAKR4) that converts the prebranch sites into a regular spacing of lateral organs. Thus, the spatiotemporal patterning of roots is fine-tuned by the root cap-specific conversion pathway of IBA to auxin and the subsequent induction of MAKR4.

International audience; 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.