Identification and Characterization of Brassinosteroid Biosynthesis and Signaling Pathway Genes in Solanum tuberosum

Russian Journal of Plant Physiology - Brassinosteroids (BRs) are plant hormones which promote plant growth and development. Their biosynthetic pathway and signal transduction pathway have been well...     

Reelin Induces Erk1/2 Signaling in Cortical Neurons Through a Non-canonical Pathway

Reelin is an extracellular protein that controls many aspects of pre- and postnatal brain development and function. The molecular mechanisms that mediate postnatal activities of Reelin are not well understood. Here, we first set out to express and purify the full length Reelin protein and a biologically active central fragment. Second, we investigated in detail the signal transduction mechanisms elicited by these purified Reelin proteins in cortical neurons. Unexpectedly, we discovered that the full-length Reelin moiety, but not the central fragment, is capable of activating Erk1/2 signaling, leading to increased p90RSK phosphorylation and the induction of immediate-early gene expression. Remarkably, Erk1/2 activation is not mediated by the canonical signal transduction pathway, involving ApoER2/VLDLR and Dab1, that mediates other functions of Reelin in early brain development. The activation of Erk1/2 signaling likely contributes to the modulation of neuronal maturation and synaptic plasticity by Reelin in the postnatal and adult brain.     

Identification of Potential Pathway Mediation Targets in Toll-like Receptor Signaling

Recent advances in reconstruction and analytical methods for signaling networks have spurred the development of large-scale models that incorporate fully functional and biologically relevant features. An extended reconstruction of the human Toll-like receptor signaling network is presented herein. This reconstruction contains an extensive complement of kinases, phosphatases, and other associated proteins that mediate the signaling cascade along with a delineation of their associated chemical reactions. A computational framework based on the methods of large-scale convex analysis was developed and applied to this network to characterize input–output relationships. The input–output relationships enabled significant modularization of the network into ten pathways. The analysis identified potential candidates for inhibitory mediation of TLR signaling with respect to their specificity and potency. Subsequently, we were able to identify eight novel inhibition targets through constraint-based modeling methods. The results of this study are expected to yield meaningful avenues for further research in the task of mediating the Toll-like receptor signaling network and its effects.     

Identification and Molecular Characterization of HOS15-interacting Proteins in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Chromatin modification is a key mechanism of gene expression in eukaryotes, and involve interactions among several proteins. Recently, we reported that HOS15, a cullin-based E3 ligase receptor, is involved in chromatin remodeling, and regulates gene expression and cold tolerance in Arabidopsis thaliana. To identify the protein complexes that function in conjunction with HOS15, we performed FLAGtag affinity purification using transgenic Arabidopsis plants expressing HOS15-FLAG, and isolated HOS15-interacting proteins. To identify these proteins, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis was conducted, and 16 proteins were identified. Database searches revealed that these proteins were histone variants, histone deacetylases, mRNA splicing regulators, a protein kinase, and proteins of unknown function. The ability of these proteins binding to HOS15 was confirmed using yeast two-hybrid, co-immunoprecipitation (Co-IP), and luciferase complementation imaging (LCI) assays. Our data suggest that specific interactions between HOS15 and those proteins involve in chromatin remodeling and RNA processing regulates plant development and abiotic stress in Arabidopsis.     

Comparing the Affinity of GTPase-binding Proteins using Competition Assays

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the binding capabilities of GTPases for two reasons: The fact that all interactions involve the same face of the GTPases means that binding events must be considered in the context of competitors, and the fact that the bound nucleotide must also be controlled means that conventional approaches such as immunoprecipitation are unsuitable for GTPase biochemistry. The assay relies on the use of purified proteins. Purified Rac1 immobilized on beads is used as the bait protein, and can be loaded with GDP, a non-hydrolyzable version of GTP or left nucleotide free, so that the signaling stage to be investigated can be controlled. The binding proteins to be investigated are purified from mammalian cells, to allow correct folding, by means of a GFP tag. Use of the same tag on both proteins is important because not only does it allow rapid purification and elution, but also allows detection of both competitors with the same antibody during elution. This means that the relative amounts of the two bound proteins can be determined accurately.     

Identification of a Functional Homolog of the Yeast Copper Homeostasis Gene ATX1 from Arabidopsis

A cDNA clone encoding a homolog of the yeast (Saccharomyces cerevisiae) gene Anti-oxidant 1 (ATX1) has been identified from Arabidopsis. This gene, referred to as Copper CHaperone (CCH), encodes a protein that is 36% identical to the amino acid sequence of ATX1 and has a 48-amino acid extension at the C-terminal end, which is absent from ATX1 homologs identified in animals. ATX1-deficient yeast (atx1) displayed a loss of high-affinity iron uptake. Expression of CCH in the atx1 strain restored high-affinity iron uptake, demonstrating that CCH is a functional homolog of ATX1. When overexpressed in yeast lacking the superoxide dismutase gene SOD1, both ATX1 and CCH protected the cell from the reactive oxygen toxicity that results from superoxide dismutase deficiency. CCH was unable to rescue the sod1 phenotype in the absence of copper, indicating that CCH function is copper dependent. In Arabidopsis CCH mRNA is present in the root, leaf, and inflorescence and is up-regulated 7-fold in leaves undergoing senescence. In plants treated with 800 nL/L ozone for 30 min, CCH mRNA levels increased by 30%. In excised leaves and whole plants treated with high levels of exogenous CuSO₄, CCH mRNA levels decreased, indicating that CCH is regulated differently than characterized metallothionein proteins in Arabidopsis.     

Computational Prediction of Candidate Proteins for S-Nitrosylation in Arabidopsis thaliana

Nitric oxide (NO) is an important signaling molecule that regulates many physiological processes in plants. One of the most important regulatory mechanisms of NO is S-nitrosylation—the covalent attachment of NO to cysteine residues. Although the involvement of cysteine S-nitrosylation in the regulation of protein functions is well established, its substrate specificity remains unknown. Identification of candidates for S-nitrosylation and their target cysteine residues is fundamental for studying the molecular mechanisms and regulatory roles of S-nitrosylation in plants. Several experimental methods that are based on the biotin switch have been developed to identify target proteins for S-nitrosylation. However, these methods have their limits. Thus, computational methods are attracting considerable attention for the identification of modification sites in proteins. Using GPS-SNO version 1.0, a recently developed S-nitrosylation site-prediction program, a set of 16,610 candidate proteins for S-nitrosylation containing 31,900 S-nitrosylation sites was isolated from the entire Arabidopsis proteome using the medium threshold. In the compartments “chloroplast,” “CUL4-RING ubiquitin ligase complex,” and “membrane” more than 70% of the proteins were identified as candidates for S-nitrosylation. The high number of identified candidates in the proteome reflects the importance of redox signaling in these compartments. An analysis of the functional distribution of the predicted candidates showed that proteins involved in signaling processes exhibited the highest prediction rate. In a set of 46 proteins, where 53 putative S-nitrosylation sites were already experimentally determined, the GPS-SNO program predicted 60 S-nitrosylation sites, but only 11 overlap with the results of the experimental approach. In general, a computer-assisted method for the prediction of targets for S-nitrosylation is a very good tool; however, further development, such as including the three dimensional structure of proteins in such analyses, would improve the identification of S-nitrosylation sites.     

Identification of Nitrogen Starvation-Responsive MicroRNAs in Arabidopsis thaliana

microRNAs (miRNAs) are a class of negative regulators that take part in many processes such as growth and development, stress responses, and metabolism in plants. Recently, miRNAs were shown to function in plant nutrient metabolism. Moreover, several miRNAs were identified in the response to nitrogen (N) deficiency. To investigate the functions of other miRNAs in N deficiency, deep sequencing technology was used to detect the expression of small RNAs under N-sufficient and -deficient conditions. The results showed that members from the same miRNA families displayed differential expression in response to N deficiency. Upon N starvation, the expression of miR169, miR171, miR395, miR397, miR398, miR399, miR408, miR827, and miR857 was repressed, whereas those of miR160, miR780, miR826, miR842, and miR846 were induced. miR826, a newly identified N-starvation-induced miRNA, was found to target the AOP2 gene. Among these N-starvation-responsive miRNAs, several were involved in cross-talk among responses to different nutrient (N, P, S, Cu) deficiencies. miR160, miR167, and miR171 could be responsible for the development of Arabidopsis root systems under N-starvation conditions. In addition, twenty novel miRNAs were identified and nine of them were significantly responsive to N-starvation. This study represents comprehensive expression profiling of N-starvation-responsive miRNAs and advances our understanding of the regulation of N homeostasis mediated by miRNAs.     

Molecular Modeling-Based Evaluation of hTLR10 and Identification of Potential Ligands in Toll-Like Receptor Signaling

Toll-like receptors (TLRs) are pattern recognition receptors that recognize pathogens based on distinct molecular signatures. The human (h)TLR1, 2, 6 and 10 belong to the hTLR1 subfamilies, which are localized in the extracellular regions and activated in response to diverse ligand molecules. Due to the unavailability of the hTLR10 crystal structure, the understanding of its homo and heterodimerization with hTLR2 and hTLR1 and the ligand responsible for its activation is limited. To improve our understanding of the TLR10 receptor-ligand interaction, we used homology modeling to construct a three dimensional (3D) structure of hTLR10 and refined the model through molecular dynamics (MD) simulations. We utilized the optimized structures for the molecular docking in order to identify the potential site of interactions between the homo and heterodimer (hTLR10/2 and hTLR10/1). The docked complexes were then used for interaction with ligands (Pam₃CSK₄ and PamCysPamSK₄) using MOE-Dock and ASEDock. Our docking studies have shown the binding orientations of hTLR10 heterodimer to be similar with other TLR2 family members. However, the binding orientation of hTLR10 homodimer is different from the heterodimer due to the presence of negative charged surfaces at the LRR11-14, thereby providing a specific cavity for ligand binding. Moreover, the multiple protein-ligand docking approach revealed that Pam₃CSK₄ might be the ligand for the hTLR10/2 complex and PamCysPamSK_4, a di-acylated peptide, might activate hTLR10/1 hetero and hTLR10 homodimer. Therefore, the current modeled complexes can be a useful tool for further experimental studies on TLR biology.     

A Trio of Viral Proteins Tunes Aphid-Plant Interactions in Arabidopsis thaliana

Background

Virus-induced deterrence to aphid feeding is believed to promote plant virus transmission by encouraging migration of virus-bearing insects away from infected plants. We investigated the effects of infection by an aphid-transmitted virus, cucumber mosaic virus (CMV), on the interaction of Arabidopsis thaliana, one of the natural hosts for CMV, with Myzus persicae (common names: ‘peach-potato aphid’, ‘green peach aphid’).

Methodology/Principal Findings

Infection of Arabidopsis (ecotype Col-0) with CMV strain Fny (Fny-CMV) induced biosynthesis of the aphid feeding-deterrent 4-methoxy-indol-3-yl-methylglucosinolate (4MI3M). 4MI3M inhibited phloem ingestion by aphids and consequently discouraged aphid settling. The CMV 2b protein is a suppressor of antiviral RNA silencing, which has previously been implicated in altering plant-aphid interactions. Its presence in infected hosts enhances the accumulation of CMV and the other four viral proteins. Another viral gene product, the 2a protein (an RNA-dependent RNA polymerase), triggers defensive signaling, leading to increased 4MI3M accumulation. The 2b protein can inhibit ARGONAUTE1 (AGO1), a host factor that both positively-regulates 4MI3M biosynthesis and negatively-regulates accumulation of substance(s) toxic to aphids. However, the 1a replicase protein moderated 2b-mediated inhibition of AGO1, ensuring that aphids were deterred from feeding but not poisoned. The LS strain of CMV did not induce feeding deterrence in Arabidopsis ecotype Col-0.

Conclusions/Significance

Inhibition of AGO1 by the 2b protein could act as a booby trap since this will trigger antibiosis against aphids. However, for Fny-CMV the interplay of three viral proteins (1a, 2a and 2b) appears to balance the need of the virus to inhibit antiviral silencing, while inducing a mild resistance (antixenosis) that is thought to promote transmission. The strain-specific effects of CMV on Arabidopsis-aphid interactions, and differences between the effects of Fny-CMV on this plant and those seen previously in tobacco (inhibition of resistance to aphids) may have important epidemiological consequences.     

Biosynthesis of Camalexin from Tryptophan Pathway Intermediates in Cell-Suspension Cultures of Arabidopsis

Camalexin (3-thiazol-2′-yl-indole) is the principal phytoalexin that accumulates in Arabidopsis after infection by fungi or bacteria. Camalexin accumulation was detectable in Arabidopsis cell-suspension cultures 3 to 5 h after inoculation with Cochliobolus carbonum (Race 1), and then increased rapidly from 7 to 24 h after inoculation. Levels of radioactivity incorporated into camalexin during a 1.5-h pulse labeling with [¹⁴C]anthranilate also increased with time after fungal inoculation. The levels of radioactive incorporation into camalexin increased rapidly between 7 and 18 h after inoculation, and then decreased along with camalexin accumulation. Relatively low levels of radioactivity from [¹⁴C]anthranilate incorporated into camalexin in the noninoculated controls. Autoradiographic analysis of the accumulation of chloroform-extractable metabolites labeled with [¹⁴C]anthranilate revealed a transient increase in the incorporation of radioactivity into indole in fungus-inoculated Arabidopsis cell cultures. The time-course measurement of radioactive incorporation into camalexin during a 1.5-h pulse labeling with [¹⁴C]indole was similar to that with [¹⁴C]anthranilate. These data suggest that indole destined for camalexin synthesis is produced by a separate enzymatic reaction that does not involve tryptophan synthase.     

The Calcium Ion Is a Second Messenger in the Nitrate Signaling Pathway of Arabidopsis

Understanding how plants sense and respond to changes in nitrogen availability is the first step toward developing strategies for biotechnological applications, such as improvement of nitrogen use efficiency. However, components involved in nitrogen signaling pathways remain poorly characterized. Calcium is a second messenger in signal transduction pathways in plants, and it has been indirectly implicated in nitrate responses. Using aequorin reporter plants, we show that nitrate treatments transiently increase cytoplasmic Ca²⁺ concentration. We found that nitrate also induces cytoplasmic concentration of inositol 1,4,5-trisphosphate. Increases in inositol 1,4,5-trisphosphate and cytoplasmic Ca²⁺ levels in response to nitrate treatments were blocked by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, a pharmacological inhibitor of phospholipase C, but not by the nonfunctional phospholipase C inhibitor analog {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}}U73343. In addition, increase in cytoplasmic Ca²⁺ levels in response to nitrate treatments was abolished in mutants of the nitrate transceptor NITRATE TRANSPORTER1.1/Arabidopsis (Arabidopsis thaliana) NITRATE TRANSPORTER1 PEPTIDE TRANSPORTER FAMILY6.3. Gene expression of nitrate-responsive genes was severely affected by pretreatments with Ca²⁺ channel blockers or phospholipase C inhibitors. These results indicate that Ca²⁺ acts as a second messenger in the nitrate signaling pathway of Arabidopsis. Our results suggest a model where NRT1.1/AtNPF6.3 and a phospholipase C activity mediate the increase of Ca²⁺ in response to nitrate required for changes in expression of prototypical nitrate-responsive genes.Plants are sessile organisms that evolved sophisticated sensing and response mechanisms to adapt to changing environmental conditions. Calcium, a ubiquitous second messenger in all eukaryotes, has been implicated in plant signaling pathways (Harper et al., 2004; Hetherington and Brownlee, 2004; Reddy and Reddy, 2004; Hepler, 2005). Multiple abiotic and biotic cues elicit specific and distinct spatiotemporal patterns of change in the concentration of cytosolic Ca²⁺ ([Ca2+]_cyt) in plants (Sanders et al., 2002; Hetherington and Brownlee, 2004; Reddy and Reddy, 2004; Hepler, 2005). Abscisic acid and heat shock treatments cause a rapid intracellular Ca²⁺ increase that is preceded by a transient increase in the level of inositol 1,4,5-trisphosphate (IP₃; Sanchez and Chua, 2001; Zheng et al., 2012). Ca²⁺ signatures are detected, decoded, and transmitted to downstream responses by a set of Ca²⁺ binding proteins that functions as Ca²⁺ sensors (White and Broadley, 2003; Dodd et al., 2010).Nitrate is the main source of N in agriculture and a potent signal that regulates the expression of hundreds of genes (Wang et al., 2004; Vidal and Gutiérrez, 2008; Ho and Tsay, 2010). Despite progress in identifying genome-wide responses, only a handful of molecular components involved in nitrate signaling has been identified. Several pieces of evidence indicate that NITRATE TRANSPORTER1.1 (NRT1.1)/Arabidopsis (Arabidopsis thaliana) NITRATE TRANSPORTER1 PEPTIDE TRANSPORTER FAMILY6.3 (AtNPF6.3) is a nitrate sensor in Arabidopsis (Ho et al., 2009; Gojon et al., 2011; Bouguyon et al., 2015). NRT1.1/AtNPF6.3 is required for normal expression of more than 100 genes in response to nitrate in Arabidopsis roots (Wang et al., 2009). Downstream of NRT1.1/AtNPF6.3, CALCINEURIN B-LIKE INTERACTING SER/THR-PROTEINE KINASE8 (CIPK8) is required for normal nitrate-induced expression of primary nitrate response genes, and the CIPK23 kinase is able to control the switch from low to high affinity of NRT1.1/AtNPF6.3 (Ho et al., 2009; Hu et al., 2009; Ho and Tsay, 2010; Castaings et al., 2011). CIPKs act in concert with CALCINEURIN B-LIKE proteins, plant-specific calcium binding proteins that activate CIPKs to phosphorylate downstream targets (Albrecht et al., 2001). Early experiments using maize (Zea mays) and barley (Hordeum vulgare) detached leaves showed that nitrate induction of two nitrate primary response genes was altered by pretreating leaves with the calcium chelator EGTA or the calcium channel blocker LaCl₃ (Sakakibara et al., 1997; Sueyoshi et al., 1999), suggesting an interplay between nitrate response and calcium-related signaling pathways. However, the role of calcium as a second messenger in the nitrate signaling pathway has not been directly addressed.We show that nitrate treatments cause a rapid increase of IP₃ and [Ca2+]_cyt levels and that blocking phospholipase C (PLC) activity inhibits both IP₃ and [Ca2+]_cyt increases after nitrate treatments. We provide evidence that NRT1.1/AtNPF6.3 is required for increasing both IP₃ and [Ca2+]_cyt in response to nitrate treatments. Altering [Ca2+]_cyt or blocking PLC activity hinders regulation of gene expression of nitrate-responsive genes. Our results indicate that Ca²⁺ is a second messenger in the nitrate signaling pathway of Arabidopsis.