The Arabidopsis root hair cell wall formation mutant lrx1 is suppressed by mutations in the RHM1 gene encoding a UDP-L-rhamnose synthase

Cell and cell wall growth are mutually dependent processes that must be tightly coordinated and controlled. LRR-extensin1 (LRX1) of Arabidopsis thaliana is a potential regulator of cell wall development, consisting of an N-terminal leucine-rich repeat domain and a C-terminal extensin-like domain typical for structural cell wall proteins. LRX1 is expressed in root hairs, and lrx1 mutant plants develop distorted root hairs that often swell, branch, or collapse. The aberrant cell wall structures found in lrx1 mutants point toward a function of LRX1 during the establishment of the extracellular matrix. To identify genes that are involved in an LRX1-dependent developmental pathway, a suppressor screen was performed on the lrx1 mutant, and two independent rol1 (for repressor of lrx1) alleles were isolated. ROL1 is allelic to Rhamnose Biosynthesis1, which codes for a protein involved in the biosynthesis of rhamnose, a major monosaccharide component of pectin. The rol1 mutations modify the pectic polysaccharide rhamnogalacturonan I and, for one allele, rhamnogalacturonan II. Furthermore, the rol1 mutations cause a change in the expression of a number of cell wall-related genes. Thus, the lrx1 mutant phenotype is likely to be suppressed by changes in pectic polysaccharides or other cell wall components.     

Synergistic interaction of the two paralogous Arabidopsis genes LRX1 and LRX2 in cell wall formation during root hair development

LRR-extensins (LRX) form a family of structural cell wall proteins containing a receptor-like domain. The functional analysis of Arabidopsis LRX1 has shown that it is involved in cell morphogenesis of root hairs. In this work, we have studied LRX2, a paralog of LRX1. LRX2 expression is mainly found in roots and is responsive to factors promoting or repressing root hair formation. The function of LRX1 and LRX2 was tested by the expression of a truncated LRX2 and different LRX1/LRX2 chimaeric proteins. Using complementation of the lrx1 phenotype as the parameter for protein function, our experiments indicate that LRX1 and LRX2 are functionally similar but show differences in their activity. Genetic analysis revealed that single lrx2 mutants do not show any defect in root hair morphogenesis, but synergistically interact with the lrx1 mutation. lrx1/lrx2 double mutants have a significantly enhanced lrx1 phenotype, resulting in frequent rupture of the root hairs soon after their initiation. Analysis of the root hair cell wall ultrastructure by transmission electron microscopy (TEM) revealed the formation of osmophilic aggregates within the wall, as well as local disintegration of the wall structure in the double mutant, but not in wild-type plants. Our results indicate that LRX1 and LRX2 have overlapping functions in root hair formation, and that they likely regulate cell morphogenesis by promoting proper development of the cell wall.     

The hydroxyproline‐rich glycoprotein domain of the Arabidopsis LRX1 requires Tyr for function but not for insolubilization in the cell wall

Extensins, hydroxyproline‐rich repetitive glycoproteins with Ser–Hyp₄ motifs, are structural proteins in plant cell walls. The leucine‐rich repeat extensin 1 (LRX1) of Arabidopsis thaliana is an extracellular protein with both a leucine‐rich repeat and an extensin domain, and has been demonstrated to be important for cell‐wall formation in root hairs. lrx1 mutants develop defective cell walls, resulting in a strong root hair phenotype. The extensin domain is essential for protein function and is thought to confer insolubilization of LRX1 in the cell wall. Here, in vivo characterization of the LRX1 extensin domain is described. First, a series of LRX1 extensin deletion constructs was produced that led to identification of a much shorter, functional extensin domain. Tyr residues can induce intra‐ and inter‐molecular cross‐links in extensins, and substitution of Tyr in the extensin domain by Phe led to reduced activity of the corresponding LRX1 protein. An additional function of Tyr (or Phe) is provided by the aromatic nature of the side chain. This suggests that these residues might be involved in hydrophobic stacking, possibly as a mechanism of protein assembly. Finally, modified LRX1 proteins lacking Tyr in the extensin domain are still insolubilized in the cell wall, indicating strong interactions of extensins within the cell wall in addition to the well‐described Tyr cross‐links.     

Fission yeast TOR signaling is essential for the down-regulation of a hyperactivated stress-response MAP kinase under salt stress

TOR (target of rapamycin) signaling regulates cell growth and division in response to environmental stimuli such as the availability of nutrients and various forms of stress. The vegetative growth of fission yeast cells, unlike other eukaryotic cells, is not inhibited by treatment with rapamycin. We found that certain mutations including pmc1Δ (Ca²⁺-ATPase), cps9-193 (small GTPase, Ryh1) and cps1-12 (1,3-β-d-glucan synthase, Bgs1) confer a rapamycin-sensitive phenotype to cells under salt stress with potassium chloride (>0.5 M). Cytometric analysis revealed that the mutant cells were unable to enter the mitotic cell cycle when treated with the drug under salt stress. Gene cloning and overexpression experiments revealed that the sensitivity to rapamycin was suppressed by the ectopic expression of tyrosine phosphatases, Pyp1 and Pyp2, which are negative regulators of Spc1/Sty1 mitogen-activated protein kinase (MAPK). The level of tyrosine phosphorylation on Spc1 was higher and sustained substantially longer in these mutants than in the wild type under salt stress. The hyperphosphorylation was significantly suppressed by overexpression of pyp1 ⁺ with concomitant resumption of the mutant cells’ growth. In fission yeast, TOR signaling has been thought to stimulate the stress-response pathway, because mutations of TORC2 components such as Tor1, Sin1 and Ste20 result in similar sensitive phenotypes to environmental stress. The present study, however, strongly suggests that TOR signaling is required for the down-regulation of a hyperactivated Spc1 for reentry into the mitotic cell cycle. This finding may shed light on our understanding of a new stress-responsive mechanism in TOR signaling in higher organisms.     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> FKBP12 binds <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> TOR and its expression in plants leads to rapamycin susceptibility

Background  

The eukaryotic TOR pathway controls translation, growth and the cell cycle in response to environmental signals such as nutrients or growth-stimulating factors. The TOR protein kinase can be inactivated by the antibiotic rapamycin following the formation of a ternary complex between TOR, rapamycin and FKBP12 proteins. The TOR protein is also found in higher plants despite the fact that they are rapamycin insensitive. Previous findings using the yeast two hybrid system suggest that the FKBP12 plant homolog is unable to form a complex with rapamycin and TOR, while the FRB domain of plant TOR is still able to bind to heterologous FKBP12 in the presence of rapamycin. The resistance to rapamycin is therefore limiting the molecular dissection of the TOR pathway in higher plants.     

The enl mutants enhance the lrx1 root hair mutant phenotype of Arabidopsis thaliana

The development of root hairs serves as an excellent model to study cell growth using both cytological and genetic approaches. In the past, we have characterized LRX1, an extracellular protein of Arabidopsis consisting of an LRR-domain and a structural extensin domain. LRX1 is specifically expressed in root hairs and lrx1 mutants show severe deficiencies in root hair development. In this work, we describe the characterization of enl (enhancer of lrx1) mutants that were isolated in a visual screen of an ethylmethanesulfonate -mutagenized lrx1 line for plants exhibiting an enhanced lrx1 phenotype. Four recessive enl mutants were analyzed, three of which define new genetic loci involved in root hair development. The mutations at the enl loci and lrx1 result in additive phenotypes in enl/lrx1 double mutants. One enl mutant is affected in the ACTIN2 gene and encodes a protein with a 22 amino acid deletion at the C-terminus. The comparison of molecular and phenotypic data of different actin2 alleles suggests that the truncated ACTIN2 protein is still partially functional.     

Growth control via TOR kinase signaling,an intracellular sensor of amino acid and energy availability,with crosstalk potential to proline metabolism

The TOR (Target of Rapamycin) protein kinase pathway plays a central role in sensing and responding to nutrients, stress, and intracellular energy state. TOR complex 1 (TORC1) is comprised of TOR, Raptor, and Lst8 and its activity is sensitive to inhibition by the macrolide antibiotic rapamycin. TORC1 regulates protein synthesis, ribosome biogenesis, autophagy, and ultimately cell growth through the phosphorylation of S6 K, 4E-BP, and other substrates. As TORC1 activity is positively or negatively modulated in response to upstream regulators, cellular growth rate is, respectively, enhanced or suppressed. A separate multiprotein TOR complex, TORC2, is insensitive to direct inhibition by rapamycin and does not regulate growth patterns directly; TORC2 can, however, impact certain aspects of TORC1 signaling and cell survival. TOR signaling is an ancient pathway, conserved among the yeasts, Dictyostelium, C. elegans, Drosophila, mammals, and Arabidopsis. This review will focus on the regulation of TORC1 in mammalian cells in the context of amino acid sensing/regulation and intracellular ATP homeostasis, but will also include comparisons among other organisms.     

Rapamycin and glucose-target of rapamycin (TOR) protein signaling in plants

Target of rapamycin (TOR) kinase is an evolutionarily conserved master regulator that integrates energy, nutrients, growth factors, and stress signals to promote survival and growth in all eukaryotes. The reported land plant resistance to rapamycin and the embryo lethality of the Arabidopsis tor mutants have hindered functional dissection of TOR signaling in plants. We developed sensitive cellular and seedling assays to monitor endogenous Arabidopsis TOR activity based on its conserved S6 kinase (S6K) phosphorylation. Surprisingly, rapamycin effectively inhibits Arabidopsis TOR-S6K1 signaling and retards glucose-mediated root and leaf growth, mimicking estradiol-inducible tor mutants. Rapamycin inhibition is relieved in transgenic plants deficient in Arabidopsis FK506-binding protein 12 (FKP12), whereas FKP12 overexpression dramatically enhances rapamycin sensitivity. The role of Arabidopsis FKP12 is highly specific as overexpression of seven closely related FKP proteins fails to increase rapamycin sensitivity. Rapamycin exerts TOR inhibition by inducing direct interaction between the TOR-FRB (FKP-rapamycin binding) domain and FKP12 in plant cells. We suggest that variable endogenous FKP12 protein levels may underlie the molecular explanation for longstanding enigmatic observations on inconsistent rapamycin resistance in plants and in various mammalian cell lines or diverse animal cell types. Integrative analyses with rapamycin and conditional tor and fkp12 mutants also reveal a central role of glucose-TOR signaling in root hair formation. Our studies demonstrate the power of chemical genetic approaches in the discovery of previously unknown and pivotal functions of glucose-TOR signaling in governing the growth of cotyledons, true leaves, petioles, and primary and secondary roots and root hairs.     

tRNA production links nutrient conditions to the onset of sexual differentiation through the TORC1 pathway

Target of rapamycin (TOR) kinase controls cell growth and metabolism in response to nutrient availability. In the fission yeast Schizosaccharomyces pombe, TOR complex 1 (TORC1) promotes vegetative growth and inhibits sexual differentiation in the presence of ample nutrients. Here, we report the isolation and characterization of mutants with similar phenotypes as TORC1 mutants, in that they initiate sexual differentiation even in nutrient‐rich conditions. In most mutants identified, TORC1 activity is downregulated and the mutated genes are involved in tRNA expression or modification. Expression of tRNA precursors decreases when cells undergo sexual differentiation. Furthermore, overexpression of tRNA precursors prevents TORC1 downregulation upon nitrogen starvation and represses the initiation of sexual differentiation. Based on these observations, we propose that tRNA precursors operate in the S. pombe TORC1 pathway to switch growth mode from vegetative to reproductive.     

TOR-mediated autophagy regulates cell death in Drosophila neurodegenerative disease

Target of rapamycin (TOR) signaling is a regulator of cell growth. TOR activity can also enhance cell death, and the TOR inhibitor rapamycin protects cells against proapoptotic stimuli. Autophagy, which can protect against cell death, is negatively regulated by TOR, and disruption of autophagy by mutation of Atg5 or Atg7 can lead to neurodegeneration. However, the implied functional connection between TOR signaling, autophagy, and cell death or degeneration has not been rigorously tested. Using the Drosophila melanogaster visual system, we show in this study that hyperactivation of TOR leads to photoreceptor cell death in an age- and light-dependent manner and that this is because of TOR''s ability to suppress autophagy. We also find that genetically inhibiting TOR or inducing autophagy suppresses cell death in Drosophila models of Huntington''s disease and phospholipase C (norpA)–mediated retinal degeneration. Thus, our data indicate that TOR induces cell death by suppressing autophagy and provide direct genetic evidence that autophagy alleviates cell death in several common types of neurodegenerative disease.     

A small G protein Rhb1 and a GTPase-activating protein Tsc2 involved in nitrogen starvation-induced morphogenesis and cell wall integrity of Candida albicans

Rheb is a new member of the small G proteins of the Ras superfamily in eukaryotic organisms and controls various physiological processes. Activity of Rheb is regulated by Tsc2, a GTPase-activating protein (GAP). In this study, we have identified Candida albicans homologs of Rheb (named as Rhb1) and Tsc2. Deletion of the RHB1 gene showed enhanced sensitivity to rapamycin (an inhibitor of TOR kinase), suggesting that Rhb1 is associated with the TOR signaling pathway in C. albicans. Further analysis indicated RHB1 and TSC2 are involved in nitrogen starvation-induced filamentation, likely by controlling the expression of MEP2 whose gene product is an ammonium permease and a sensor for the nitrogen signal. Moreover, we have demonstrated that Rhb1 is also involved in cell wall integrity pathway, by transferring signals through the TOR kinase and the Mkc1 MAP kinase pathway. Together, this study brings new insights into the complex interplay of signaling and regulatory pathways in C. albicans.     

Phosphatase-associated protein MoTip41 interacts with the phosphatase MoPpe1 to mediate crosstalk between TOR and cell wall integrity signalling during infection by the rice blast fungus Magnaporthe oryzae

The type 2A (PP2A) and type 2A-like (PP4 and PP6) serine/threonine phosphatases participate in a variety of cellular processes, such as cell cycle progression, signal transduction and apoptosis. Previously, we reported that the PP6 catalytic subunit MoPpe1, which interacts with and is suppressed by type 2A associated protein of 42 kDa (MoTap42), an essential protein involved in the target of rapamycin (TOR) signalling pathway, has important roles in development, virulence and activation of the cell wall integrity (CWI) pathway in the rice blast fungus Magnaporthe oryzae. Here, we show that Tap42-interacting protein 41 (MoTip41) mediates crosstalk between the TOR and CWI signalling pathways; and participates in the TOR pathway via interaction with MoPpe1, but not MoTap42. The deletion of MoTIP41 resulted in disruption of CWI signalling, autophagy, vegetative growth, appressorium function and plant infection, as well as increased sensitivity to rapamycin. Further investigation revealed that MoTip41 modulates activation of the CWI pathway in response to infection by interfering with the interaction between MoTap42 and MoPpe1. These findings enhance our understanding of how crosstalk between TOR and CWI signalling modulates the development and pathogenicity of M. oryzae.     

Identification and characterization of PP2C phosphatase SjPtc1 in Schistosoma japonicum

Protein phosphorylation, regulated by protein kinases and protein phosphatases, is crucial for protein structure and function in eukaryotic organisms. Type 2C protein phosphatase (PP2C) belongs to the serine/threonine phosphatase family and its activities require the presence of a divalent magnesium or manganese ion. In the present study, a potential PP2C phosphatase (SjPtc1) was identified in Schistosoma japonicum. The SjPTC1 gene was found to be highly expressed in adult worms. A recombinant SjPtc1 protein showed typical PP2C phosphatase activity. Heterologous SjPTC1 expression reversed the sensitivity of yeast ptc1 null mutants toward H₂O₂, ZnCl₂, cisplatin, and rapamycin. Collectively, the results suggest that SjPtc1 may take part in the regulation of cellular responses to oxidative stress, DNA damage stress, and the TOR (target of rapamycin) signaling pathway.     

Cyclin G Functions as a Positive Regulator of Growth and Metabolism in Drosophila

In multicellular organisms, growth and proliferation is adjusted to nutritional conditions by a complex signaling network. The Insulin receptor/target of rapamycin (InR/TOR) signaling cascade plays a pivotal role in nutrient dependent growth regulation in Drosophila and mammals alike. Here we identify Cyclin G (CycG) as a regulator of growth and metabolism in Drosophila. CycG mutants have a reduced body size and weight and show signs of starvation accompanied by a disturbed fat metabolism. InR/TOR signaling activity is impaired in cycG mutants, combined with a reduced phosphorylation status of the kinase Akt1 and the downstream factors S6-kinase and eukaryotic translation initiation factor 4E binding protein (4E-BP). Moreover, the expression and accumulation of Drosophila insulin like peptides (dILPs) is disturbed in cycG mutant brains. Using a reporter assay, we show that the activity of one of the first effectors of InR signaling, Phosphoinositide 3-kinase (PI3K92E), is unaffected in cycG mutants. However, the metabolic defects and weight loss in cycG mutants were rescued by overexpression of Akt1 specifically in the fat body and by mutants in widerborst (wdb), the B''-subunit of the phosphatase PP2A, known to downregulate Akt1 by dephosphorylation. Together, our data suggest that CycG acts at the level of Akt1 to regulate growth and metabolism via PP2A in Drosophila.