Fatty acid-derived signals in plants

Plants synthesize many fatty acid derivatives, several of which play important regulatory roles. Jasmonates are the best characterized examples. Jasmonate-insensitive mutants and mutants with a constitutive jasmonate response have given us new insights into jasmonate signalling. The jasmonate biosynthesis mutant opr3 allowed the dissection of cyclopentanone and cyclopentenone signalling, thus defining specific roles for these molecules. Jasmonate signalling is a complex network of individual signals and recent findings on specific activities of methyl jasmonate and (Z)-jasmone add to this picture. In addition, there are keto, hydroxy and hydroperoxy fatty acids that might be involved in cell death and the expression of stress-related genes. Finally, there are bruchins and volicitin, signal molecules from insects that are perceived by plants in the picomole to femtomole range. They highlight the importance of fatty acid-derived molecules in interspecies communication and in plant defence.     

Inhibition of tumor necrosis factor-induced phenotypes by short intracellular versions of latent membrane protein-1

Tumor necrosis factor (TNF) is a potent multi-functional cytokine with a homeostatic role in host defence. In case of deregulation, TNF is implicated in numerous pathologies. The latent membrane protein-1 (LMP1) is expressed by Epstein–Barr virus during viral latency and displaying properties of a constitutively activated member of the TNF receptor family. Both TNFR1 and LMP1 share a similar set of proximal adapters and signalling pathways although they display different biological responses. We previously demonstrated that the intracellular part of LMP1, LMP1-CT, a dominant-negative form of LMP1, inhibits LMP1 signalling.Here, we developed shorter versions derived from C-terminal part of LMP1 to investigate their roles on LMP1 and TNF signalling. We constructed several mutants of LMP1 containing a part of cytoplasmic signalling region fused to the green fluorescent protein. These mutants selectively impair signalling by LMP1 and TNF but not by IL-1β which uses other adapters. Dominant-negative effect was due to binding and sequestration of LMP1 adapters RIP, TRAF2 and TRADD as assessed by coimmunoprecipitation experiments and confocal analysis. Expression of these mutants impairs the recruitment of these adapters by TNFR1 and TNF-associated phenotypes. These mutants did not display cytostatic properties but were able to modulate TNF-induced phenotypes, apoptosis or cell survival, depending on the cell context. Interestingly, these mutants are able to inhibit a pro-inflammatory response in endothelial cells. These data demonstrate that LMP1 derived molecules can be used to design compounds with potential therapeutic roles in diseases due to TNF overactivation.     

Lipid function in plant cell polarity

The establishment and maintenance of cell polarity play pivotal roles during plant development. During the past five years, proteins that are required for different aspects of plant cell polarity have been identified. However, the functions of lipids and their interactions with proteins that mediate polarity remained largely unaddressed. Recent genetic studies have discovered cell and tissue polarity mutants that have defects in sterol composition, glycosylphosphatidylinositol-anchored proteins, glycosylphosphatidylinositol biosynthesis and phospholipid signalling. Analyses of the affected gene products have provided a first glance at the roles of lipids in cell polarity signalling, as well as in the trafficking and anchoring of polar proteins.     

Echinoid limits R8 photoreceptor specification by inhibiting inappropriate EGF receptor signalling within R8 equivalence groups

EGF receptor signalling plays diverse inductive roles during development. To achieve this, its activity must be carefully regulated in a variety of ways to control the time, pattern, intensity and duration of signalling. We show that the cell surface protein Echinoid is required to moderate Egfr signalling during R8 photoreceptor selection by the proneural gene atonal during Drosophila eye development. In echinoid mutants, Egfr signalling is increased during R8 formation, and this causes isolated R8 cells to be replaced by groups of two or three cells. This mutant phenotype resembles the normal inductive function of Egfr in other developmental contexts, particularly during atonal-controlled neural recruitment of chordotonal sense organ precursors. We suggest that echinoid acts to prevent a similar inductive outcome of Egfr signalling during R8 selection.     

The Shh signalling pathway in early tooth development.

The Sonic Hedgehog (Shh) signalling pathway has been proposed to play an important role in mammalian tooth development. We describe the spatial and temporal expression of genes in this pathway during early tooth development and interpret these patterns in terms of the likely roles of Shh signalling. We show that the two putative receptors of the Shh ligand, Ptc and Ptch-2, localise in different cells, suggesting Shh may function in different ways as an epithelial and mesenchymal signal. Shh signalling has previously been shown, in other organs, to stimulate cell proliferation. In this paper we analyse the Fgf signalling pathway in Gli-2 mutants and propose a mechanism as to how Gli-2 may regulate cell proliferation in tooth development.     

Suppression of auxin signalling promotes rice susceptibility to Rice black streaked dwarf virus infection

Auxin plays a fundamental role in plant growth and development, and also influences plant defence against various pathogens. Previous studies have examined the different roles of the auxin pathway during infection by biotrophic bacteria and necrotrophic fungi. We now show that the auxin signalling pathway was markedly down-regulated following infection of rice by Rice black streaked dwarf virus (RBSDV), a dsRNA virus. Repression of the auxin receptor TIR1 by a mutant overexpressing miR393 increased rice susceptibility to RBSDV. Mutants overexpressing the auxin signalling repressors OsIAA20 and OsIAA31 were also more susceptible to RBSDV. The induction of jasmonic acid (JA) pathway genes in response to RBSDV was supressed in auxin signalling mutants, suggesting that activation of the JA pathway may be part of the auxin signalling-mediated rice defence against RBSDV. More importantly, our results also revealed that OsRboh-mediated reactive oxygen species levels played important roles in this defence. The results offer novel insights into the regulatory mechanisms of auxin signalling in the rice–RBSDV interaction.     

Polarity and signalling in plant embryogenesis

The establishment of the apical-basal axis is a critical event in plant embryogenesis, evident from the earliest stages onwards. Polarity is evident in the embryo sac, egg cell, zygote, and embryo-suspensor complex. In the embryo-proper, two functionally distinct meristems form at each pole, through the localized expression of key genes. A number of mutants, notably of the model genetic organism Arabidopsis thaliana, have revealed new gene functions that are required for patterning of the apical-basal axis. There is now increasing evidence that two particular modes of signalling, via auxin and cell wall components, play important roles in co-ordinating the gene expression programmes that define determinative roles in the establishment of polarity.     

OipA plays a role in Helicobacter pylori-induced focal adhesion kinase activation and cytoskeletal re-organization

The initial signalling events leading to Helicobacter pylori infection associated changes in motility, cytoskeletal reorganization and elongation of gastric epithelial cells remain poorly understood. Because focal adhesion kinase (FAK) is known to play important roles in regulating actin cytoskeletal organization and cell motility we examined the effect of H. pylori in gastric epithelial cells co-cultured with H. pylori or its isogenic cag pathogenicity island (PAI) or oipA mutants. H. pylori induced FAK phosphorylation at distinct tyrosine residues in a dose- and time-dependent manner. Autophosphorylation of FAK Y397 was followed by phosphorylation of Src Y418 and resulted in phosphorylation of the five remaining FAK tyrosine sites. Phosphorylated FAK and Src activated Erk and induced actin stress fibre formation. FAK knock-down by FAK-siRNA inhibited H. pylori- mediated Erk phosphorylation and abolished stress fibre formation. Infection with oipA mutants reduced phosphorylation of Y397, Y576, Y577, Y861 and Y925, inhibited stress fibre formation and altered cell morphology. cag PAI mutants reduced phosphorylation of only FAK Y407 and had less effect on stress fibre formation than oipA mutants. We propose that activation of FAK and Src are responsible for H. pylori -induced induction of signalling pathways resulting in the changes in cell phenotype important for pathogenesis.     

Mammalian Rho GTPases: new insights into their functions from in vivo studies

Rho GTPases are key regulators of cytoskeletal dynamics and affect many cellular processes, including cell polarity, migration, vesicle trafficking and cytokinesis. These proteins are conserved from plants and yeast to mammals, and function by interacting with and stimulating various downstream targets, including actin nucleators, protein kinases and phospholipases. The roles of Rho GTPases have been extensively studied in different mammalian cell types using mainly dominant negative and constitutively active mutants. The recent availability of knockout mice for several members of the Rho family reveals new information about their roles in signalling to the cytoskeleton and in development.     

Arabidopsis mutants of AtABCG22, an ABC transporter gene, increase water transpiration and drought susceptibility

In plants, water vapour is released into the atmosphere through stomata in a process called transpiration. Abscisic acid (ABA) is a key phytohormone that facilitates stomatal closure through its action on guard cells. Recently, ATP-binding cassette (ABC) transporter genes, AtABCG25 and AtABCG40, were shown to be involved in ABA transport and responses. However, the functions of many other AtABCG family genes are still unknown. Here, we identified another ABCG gene (AtABCG22) that is required for stomatal regulation in Arabidopsis. The atabcg22 mutant plants had lower leaf temperatures and increased water loss, implying elevated transpiration through an influence on stomatal regulation. We also found that atabcg22 plants were more suspectible to drought stress than wild-type plants. AtABCG22 was expressed in aerial organs, mainly guard cells, in which the gene expression pattern was consistent with the mutant phenotypes. Using double mutants, we investigated the genetic relationships between the mutations. The atabcg22 mutation further increased the water loss of srk2e/ost1 mutants, which were defective in ABA signalling in guard cells. Also, the atabcg22 mutation enhanced the phenotype of nced3 mutants, which were defective in ABA biosynthesis. Accordingly, the additive roles of AtABCG22 functions in ABA signalling and ABA biosynthesis are discussed.     

Distinct and collaborative roles of Drosophila EXT family proteins in morphogen signalling and gradient formation

Heparan sulfate proteoglycans (HSPG) have been implicated in regulating the signalling activities of secreted morphogen molecules including Wingless (Wg), Hedgehog (Hh) and Decapentaplegic (Dpp). HSPG consists of a protein core to which heparan sulfate (HS) glycosaminoglycan (GAG) chains are attached. The formation of HS GAG chains is catalyzed by glycosyltransferases encoded by members of the EXT family of putative tumor suppressors linked to hereditary multiple exostoses. Previous studies in Drosophila demonstrated that tout-velu (ttv), the Drosophila EXT1, is required for Hh movement. However, the functions of other EXT family members are unknown. We have identified and isolated the other two members of the Drosophila EXT family genes, which are named sister of tout-velu (sotv) and brother of tout-velu (botv), and encode Drosophila homologues of vertebrate EXT2 and EXT-like 3 (EXTL3), respectively. We show that both Hh and Dpp signalling activities, as well as their morphogen distributions, are defective in cells mutant for ttv, sotv or botv in the wing disc. Surprisingly, although Wg morphogen distribution is abnormal in ttv, sotv and botv, Wg signalling is only defective in botv mutants or ttv-sotv double mutants, and not in ttv nor sotv alone, suggesting that Ttv and Sotv are redundant in Wg signalling. We demonstrate further that Ttv and Sotv form a complex and are co-localized in vivo. Our results, along with previous studies on Ttv, provide evidence that all three Drosophila EXT proteins are required for the biosynthesis of HSPGs, and for the gradient formation of the Wg, Hh and Dpp morphogens. Our results also suggest that HSPGs have two distinct roles in Wg morphogen distribution and signalling.     

HPS4/SABRE regulates plant responses to phosphate starvation through antagonistic interaction with ethylene signalling

The phytohormone ethylene plays important roles in regulating plant responses to phosphate (Pi) starvation. To date, however, no molecular components have been identified that interact with ethylene signalling in regulating such responses. In this work, an Arabidopsis mutant, hps4, was characterized that exhibits enhanced responses to Pi starvation, including increased inhibition of primary root growth, enhanced expression of Pi starvation-induced genes, and overproduction of root-associated acid phosphatases. Molecular cloning indicated that hps4 is a new allele of SABRE, which was previously identified as an important regulator of cell expansion in Arabidopsis. HPS4/SABRE antagonistically interacts with ethylene signalling to regulate plant responses to Pi starvation. Furthermore, it is shown that Pi-starved hps4 mutants accumulate more auxin in their root tips than the wild type, which may explain the increased inhibition of their primary root growth when grown under Pi deficiency.     

Regulation of basal and oxidative stress‐triggered jasmonic acid‐related gene expression by glutathione

Glutathione is a determinant of cellular redox state with roles in defence and detoxification. Emerging concepts suggest that this compound also has functions in cellular signalling. Here, we report evidence that glutathione plays potentially important roles in setting signalling strength through the jasmonic acid (JA) pathway. Firstly, we show that basal expression of JA‐related genes is correlated with leaf glutathione content when the latter is manipulated either genetically or pharmacologically. Secondly, analyses of an oxidative stress signalling mutant, cat2, reveal that up‐regulation of the JA pathway triggered by intracellular oxidation requires accompanying glutathione accumulation. Genetically blocking this accumulation in a cat2 cad2 line largely annuls H₂O₂‐induced expression of JA‐linked genes, and this effect can be rescued by exogenously supplying glutathione. While most attention on glutathione functions in biotic stress responses has been focused on the thiol‐regulated protein NPR1, a comparison of JA‐linked gene expression in cat2 cad2 and cat2 npr1 double mutants provides evidence that glutathione acts through other components to regulate the response of this pathway to oxidative stress. Our study provides new information implicating glutathione as a factor determining basal JA gene expression and suggests novel glutathione‐dependent control points that regulate JA signalling in response to intracellular oxidation.     

The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling pathway

The Arabidopsis ABI1 and ABI2 genes encode two protein serine/threonine phosphatases 2C (PP2C). These genes have been originally identified by the dominant mutations abi1--1 and abi2--1, which reduce the plant's responsiveness to the hormone abscisic acid (ABA). However, recessive mutants of ABI1 were recently shown to be supersensitive to ABA, which demonstrated that the ABI1 phosphatase is a negative regulator of ABA signalling. We report here the isolation and characterisation of the first reduction-of-function allele of ABI2, abi2--1R1. The in vitro phosphatase activity of the abi2--1R1 protein is approximately 100-fold lower than that of the wild-type ABI2 protein. Abi2--1R1 plants displayed a wild-type ABA sensitivity. However, doubly mutant plants combining the abi2--1R1 allele and a loss-of-function allele at the ABI1 locus were more responsive to ABA than each of the parental single mutants. These data indicate that the wild-type ABI2 phosphatase is a negative regulator of ABA signalling, and that the ABI1 and ABI2 phosphatases have overlapping roles in controlling ABA action. Measurements of PP2C activity in plant extracts showed that the phosphatase activity of ABI1 and ABI2 increases in response to ABA. These results suggest that ABI1 and ABI2 act in a negative feedback regulatory loop of the ABA signalling pathway.     

HSPG synthesis by zebrafish Ext2 and Extl3 is required for Fgf10 signalling during limb development

Heparan sulphate proteoglycans (HSPGs) are known to be crucial for signalling by the secreted Wnt, Hedgehog, Bmp and Fgf proteins during invertebrate development. However, relatively little is known about their effect on developmental signalling in vertebrates. Here, we report the analysis of daedalus, a novel zebrafish pectoral fin mutant. Positional cloning identified fgf10 as the gene disrupted in daedalus. We find that fgf10 mutants strongly resemble zebrafish ext2 and extl3 mutants, which encode glycosyltransferases required for heparan sulphate biosynthesis. This suggests that HSPGs are crucial for Fgf10 signalling during limb development. Consistent with this proposal, we observe a strong genetic interaction between fgf10 and extl3 mutants. Furthermore, application of Fgf10 protein can rescue target gene activation in fgf10, but not in ext2 or extl3 mutants. By contrast, application of Fgf4 protein can activate target genes in both ext2 and extl3 mutants, indicating that ext2 and extl3 are differentially required for Fgf10, but not Fgf4, signalling during limb development. This reveals an unexpected specificity of HSPGs in regulating distinct vertebrate Fgfs.