Focus on Ethylene: Role of Ethylene and Its Cross Talk with Other Signaling Molecules in Plant Responses to Heavy Metal Stress

Excessive heavy metals (HMs) in agricultural lands cause toxicities to plants, resulting in declines in crop productivity. Recent advances in ethylene biology research have established that ethylene is not only responsible for many important physiological activities in plants but also plays a pivotal role in HM stress tolerance. The manipulation of ethylene in plants to cope with HM stress through various approaches targeting either ethylene biosynthesis or the ethylene signaling pathway has brought promising outcomes. This review covers ethylene production and signal transduction in plant responses to HM stress, cross talk between ethylene and other signaling molecules under adverse HM stress conditions, and approaches to modify ethylene action to improve HM tolerance. From our current understanding about ethylene and its regulatory activities, it is believed that the optimization of endogenous ethylene levels in plants under HM stress would pave the way for developing transgenic crops with improved HM tolerance.In addition to common abiotic stresses seen in agricultural production, such as drought, submerging, and extreme temperatures (Thao and Tran, 2012; Xia et al., 2015), heavy metal (HM) stress has arisen as a new pervasive threat (Srivastava et al., 2014; Ahmad et al., 2015). This is mainly due to the unrestricted industrialization and urbanization carried out during the past few decades, which have led to the increase of HMs in soils. Plants naturally require more than 15 different types of HM as nutrients serving for biological activities in cells (Sharma and Chakraverty, 2013). However, when the nutritional/nonnutritional HMs are present in excess, plants have to either suffer or take these up from the soil in an unwilling manner (Nies, 1999; Sharma and Chakraverty, 2013). Upon HM stress exposure, plants induce oxidative stress due to the excessive production of reactive oxygen species (ROS) and methylglyoxal (Sharma and Chakraverty, 2013). High levels of these compounds have been shown to negatively affect cellular structure maintenance (e.g. induction of lipid peroxidation in the membrane, biological macromolecule deterioration, ion leakage, and DNA strand cleavage; Gill and Tuteja, 2010; Nagajyoti et al., 2010) as well as many other biochemical and physiological processes (Dugardeyn and Van Der Straeten, 2008). As a result, plant growth is retarded and, ultimately, economic yield is decreased (Yadav, 2010; Anjum et al., 2012; Hossain et al., 2012; Asgher et al., 2015). Moreover, the accumulation of metal residues in the major food chain has been shown to cause serious ecological and health problems (Malik, 2004; Verstraeten et al., 2008).Plants employ different strategies to detoxify the unwanted HMs. Among the common responses of plants to HM stress are increases in ethylene production due to the enhanced expression of ethylene-related biosynthetic genes (Asgher et al., 2014; Khan and Khan, 2014; Khan et al., 2015b) and/or changes in the expression of ethylene-responsive genes (Maksymiec, 2007). Conventionally, this hormone has been established to modulate a number of important plant physiological activities, including seed germination, root hair and root nodule formation, and maturation (fruit ripening in particular; Dugardeyn and Van Der Straeten, 2008). On the other hand, although ethylene has also been suggested to be a stress-related hormone responding to a number of biotic and abiotic triggers, little is known about the exact role of elevated HM stress-related ethylene in plants (Zapata et al., 2003). Enhanced production of ethylene in plants subjected to toxic levels of cadmium (Cd), copper (Cu), iron (Fe), nickel (Ni), and zinc (Zn) has been shown (Maksymiec, 2007). As an example, Cd- and Cu-mediated stimulation of ethylene synthesis has been reported as a result of the increase of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) activity, one of the enzymes involved in the ethylene synthesis pathway (Schlagnhaufer and Arteca, 1997; Khan et al., 2015b).Plants tend to adjust or induce adaptation or tolerance mechanisms to overcome stress conditions. To develop stress tolerance, plants trigger a network of hormonal cross talk and signaling, among which ethylene production and signaling are prominently involved in stress-induced symptoms in acclimation processes (Gazzarrini and McCourt, 2003). Therefore, the necessity of controlling ethylene homeostasis and signal transduction using biochemical and molecular tools remains open to combat stress situations. Stress-induced ethylene acts to trigger stress-related effects on plants because of the autocatalytic ethylene synthesis. Autocatalytic stress-related ethylene production is controlled by mitogen-activated protein kinase (MAPK) phosphorylation cascades (Takahashi et al., 2007) and through stabilizing ACS2/6 (Li et al., 2012). Strong lines of evidence have shown the multiple facets of ethylene in plant responses to different abiotic stresses, including excessive HM, depending upon endogenous ethylene concentration and ethylene sensitivities that differ in developmental stage, plant species, and culture systems (Pierik et al., 2006; Kim et al., 2008; Khan and Khan, 2014). Under HM stress conditions, plants show a rapid increase in ethylene production and reduced plant growth and development, suggesting a negative regulatory role of ethylene in plant responses to HM stress (Schellingen et al., 2014; Khan et al., 2015b). On the other hand, a potential involvement of ETHYLENE INSENSITIVE2 (EIN2), a central component of the ethylene signaling pathway, as a positive regulator in lead (Pb) resistance in Arabidopsis (Arabidopsis thaliana) has also been demonstrated (Cao et al., 2009). More recently, Khan and Khan (2014) showed that ethylene-regulated antioxidant metabolism maintained a higher level of reduced glutathione (GSH) and alleviated photosynthetic inhibition in mustard (Brassica juncea) plants exposed to Ni, Zn, or Cd through the optimization of ethylene homeostasis (Masood et al., 2012). Taken together, the purpose of this review is to update the research community with our current understanding of the roles of ethylene and its signaling in plant responses to HM stress. Moreover, the cross talk of ethylene with other phytohormones and signaling molecules upon HM stress will also be discussed.     

Genetic link between Cabeza, a Drosophila homologue of Fused in Sarcoma (FUS), and the EGFR signaling pathway

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive muscular weakness. Fused in Sarcoma (FUS) that has been identified in familial ALS is an RNA binding protein that is normally localized in the nucleus. However, its function in vivo is not fully understood. Drosophila has Cabeza (Caz) as a FUS homologue and specific knockdown of Caz in the eye imaginal disc and pupal retina using a GMR-GAL4 driver was here found to induce an abnormal morphology of the adult compound eyes, a rough eye phenotype. This was partially suppressed by expression of the apoptosis inhibitor P35. Knockdown of Caz exerted no apparent effect on differentiation of photoreceptor cells. However, immunostaining with an antibody to Cut that marks cone cells revealed fusion of these and ommatidia of pupal retinae. These results indicate that Caz knockdown induces apoptosis and also inhibits differentiation of cone cells, resulting in abnormal eye morphology in adults. Mutation in EGFR pathway-related genes, such as rhomboid-1, rhomboid-3 and mirror suppressed the rough eye phenotype induced by Caz knockdown. Moreover, the rhomboid-1 mutation rescued the fusion of cone cells and ommatidia observed in Caz knockdown flies. The results suggest that Caz negatively regulates the EGFR signaling pathway required for determination of cone cell fate in Drosophila.     

Single Nucleotide Polymorphisms of One-Carbon Metabolism and Cancers of the Esophagus,Stomach, and Liver in a Chinese Population

One-carbon metabolism (folate metabolism) is considered important in carcinogenesis because of its involvement in DNA synthesis and biological methylation reactions. We investigated the associations of single nucleotide polymorphisms (SNPs) in folate metabolic pathway and the risk of three GI cancers in a population-based case-control study in Taixing City, China, with 218 esophageal cancer cases, 206 stomach cancer cases, 204 liver cancer cases, and 415 healthy population controls. Study participants were interviewed with a standardized questionnaire, and blood samples were collected after the interviews. We genotyped SNPs of the MTHFR, MTR, MTRR, DNMT1, and ALDH2 genes, using PCR-RFLP, SNPlex, or TaqMan assays. To account for multiple comparisons and reduce the chances of false reports, we employed semi-Bayes (SB) shrinkage analysis. After shrinkage and adjusting for potential confounding factors, we found positive associations between MTHFR rs1801133 and stomach cancer (any T versus C/C, SB odds-ratio [SBOR]: 1.79, 95% posterior limits: 1.18, 2.71) and liver cancer (SBOR: 1.51, 95% posterior limits: 0.98, 2.32). There was an inverse association between DNMT1 rs2228612 and esophageal cancer (any G versus A/A, SBOR: 0.60, 95% posterior limits: 0.39, 0.94). In addition, we detected potential heterogeneity across alcohol drinking status for ORs relating MTRR rs1801394 to esophageal (posterior homogeneity P = 0.005) and stomach cancer (posterior homogeneity P = 0.004), and ORs relating MTR rs1805087 to liver cancer (posterior homogeneity P = 0.021). Among non-alcohol drinkers, the variant allele (allele G) of these two SNPs was inversely associated with the risk of these cancers; while a positive association was observed among ever-alcohol drinkers. Our results suggest that genetic polymorphisms related to one-carbon metabolism may be associated with cancers of the esophagus, stomach, and liver. Heterogeneity across alcohol consumption status of the associations between MTR/MTRR polymorphisms and these cancers indicates potential interactions between alcohol drinking and one-carbon metabolic pathway.     

Mannose-binding lectin gene polymorphisms and hepatitis B virus infection in Vietnamese patients

Mannose-binding lectin (MBL) is a constituent of the human innate immune system which may play an important role in combating a variety of infectious diseases. We investigated the distribution of MBL gene mutations in a Vietnamese population, using polymerase chain reaction and DNA sequence analysis, and sought associations with the outcome of hepatitis B virus (HBV) infection. For this purpose we used samples from a total of 123 patients with confirmed, well-defined HBV infections, representing a full spectrum of clinical presentation from acute to chronic to malignant states, as well as from 112 healthy controls. The only MBL gene mutation found in this population, that at codon 54 of exon 1, was present at an overall frequency of 0.12, with a trend towards a higher frequency in the HBV-infected group compared with controls (0.15 versus 0.08, P = 0.079). Within the HBV-infected group there was a non-significant trend towards higher viral loads in those with this mutation, accompanied by significantly higher serum transaminase levels in the same individuals. Segregation according to clinical presentation showed that the mutation was present at a significantly higher frequency in the group with acute hepatitis B (AHB) compared with the healthy control group (0.25 versus 0.08, P = 0.01), and was associated with higher serum transaminase levels. Our results indicate that a mutation of the MBL gene might influence the clinical outcome of HBV infection in Vietnamese patients.     

MRSA epidemic linked to a quickly spreading colonization and virulence determinant

The molecular processes underlying epidemic waves of methicillin-resistant Staphylococcus aureus (MRSA) infection are poorly understood(1). Although a major role has been attributed to the acquisition of virulence determinants by horizontal gene transfer(2), there are insufficient epidemiological and functional data supporting that concept. We here report the spread of clones containing a previously extremely rare(3,4) mobile genetic element–encoded gene, sasX. We demonstrate that sasX has a key role in MRSA colonization and pathogenesis, substantially enhancing nasal colonization, lung disease and abscess formation and promoting mechanisms of immune evasion. Moreover, we observed the recent spread of sasX from sequence type 239 (ST239) to invasive clones belonging to other sequence types. Our study identifies sasX as a quickly spreading crucial determinant of MRSA pathogenic success and a promising target for therapeutic interference. Our results provide proof of principle that horizontal gene transfer of key virulence determinants drives MRSA epidemic waves.     

Individual variations in arsenic metabolism in Vietnamese: the association with arsenic exposure and GSTP1 genetic polymorphism

We investigated the association of As exposure and genetic polymorphism in glutathione S-transferase π1 (GSTP1) with As metabolism in 190 local residents from the As contaminated groundwater areas in the Red River Delta, Vietnam. Total As concentrations in groundwater ranged from <0.1 to 502 μg l(-1). Concentrations of dimethylarsinic acid (DMA(V)), monomethylarsonic acid (MMA(V)), and arsenite (As(III)) in human urine were positively correlated with total As levels in the groundwater, suggesting that people in these areas may be exposed to As through the groundwater. The concentration ratios of urinary As(III)/arsenate (As(V)) and MMA(V)/inorganic As (IA; As(III) + As(V))(M/I), which are indicators of As metabolism, increased with the urinary As level. Concentration and proportion of As(III) were high in the wild type of GSTP1 Ile105Val compared with the hetero type, and these trends were more pronounced in the higher As exposure group (>56 μg l(-1) creatinine in urine), but not in the lower exposure group. In the high As exposure group, As(III)/As(V) ratios in the urine of wild type of GSTP1 Ile105Val were significantly higher than those of the hetero type, while the opposite trend was observed for M/I. These results suggest that the excretion and metabolism of IA may depend on both the As exposure level and the GSTP1 Ile105Val genotype.     

Modifying the N-terminus of polyamides: PyImPyIm has improved sequence specificity over f-ImPyIm

Seven N-terminus modified derivatives of a previously published minor-groove binding polyamide (f-ImPyIm, 1) were synthesized and the biochemical and biophysical chemistry evaluated. These compounds were synthesized with the aim of attaining a higher level of sequence selectivity over f-ImPyIm (1), a previously published strong minor-groove binder. Two compounds possessing a furan or a benzofuran moiety at the N-terminus showed a footprint of 0.5 μM at the cognate ACGCGT site (determined by DNase I footprinting); however, the specificity of these compounds was not improved. In contrast, PyImPyIm (4) produced a footprint of 0.5 μM but showed a superior specificity using the same technique. When evaluated by thermal melting experiments and circular dichroism using ACGCGT and the non-cognate AAATTT sequence, all compounds were shown to bind in the minor-groove of DNA and stabilize the cognate sequence much better than the non-cognate (except for the non-amido-compound that did not bind either sequence, as expected). PyImPyIm (4) was interesting as the ΔT_m for this compound was only 4 °C but the footprint was very selective. No binding was observed for this compound with a third DNA (non-cognate, ACCGGT). ITC studies on compound 4 showed exothermic binding with ACGCGT and no heat change was observed for titrating the compound to the other two DNA sequences. The heat capacity (ΔC_p) of the PIPI/ACGCGT complex calculated from the hydrophobic interactions and SASA calculations was comparable to the experimental value obtained from ITC (−146 cal mol⁻¹ K⁻¹). SPR results provided confirmation of the sequence specificity of PyImPyIm (4), with a K_eq value determined to be 7.1 × 10⁶ M⁻¹ for the cognate sequence and no observable binding to AAATTT and ACCGGT. Molecular dynamic simulations affirmed that PyImPyIm (4) binds as a dimer in an overlapped conformation, and it fits snugly in the minor-groove of the ACGCGT oligonucleotide. PyImPyIm (4) is an especially interesting molecule, because although the binding affinity is slightly reduced, the specificity with respect to f-ImPyIm (1) is significantly improved.     

A multi-center randomised controlled trial of gatifloxacin versus azithromycin for the treatment of uncomplicated typhoid fever in children and adults in Vietnam

Background

Drug resistant typhoid fever is a major clinical problem globally. Many of the first line antibiotics, including the older generation fluoroquinolones, ciprofloxacin and ofloxacin, are failing.

Objectives

We performed a randomised controlled trial to compare the efficacy and safety of gatifloxacin (10 mg/kg/day) versus azithromycin (20 mg/kg/day) as a once daily oral dose for 7 days for the treatment of uncomplicated typhoid fever in children and adults in Vietnam.

Methods

An open-label multi-centre randomised trial with pre-specified per protocol analysis and intention to treat analysis was conducted. The primary outcome was fever clearance time, the secondary outcome was overall treatment failure (clinical or microbiological failure, development of typhoid fever-related complications, relapse or faecal carriage of S. typhi).

Principal Findings

We enrolled 358 children and adults with suspected typhoid fever. There was no death in the study. 287 patients had blood culture confirmed typhoid fever, 145 patients received gatifloxacin and 142 patients received azithromycin. The median FCT was 106 hours in both treatment arms (95% Confidence Interval [CI]; 94–118 hours for gatifloxacin versus 88–112 hours for azithromycin), (logrank test p = 0.984, HR [95% CI] = 1.0 [0.80–1.26]).Overall treatment failure occurred in 13/145 (9%) patients in the gatifloxacin group and 13/140 (9.3%) patients in the azithromycin group, (logrank test p = 0.854, HR [95% CI] = 0.93 [0.43–2.0]). 96% (254/263) of the Salmonella enterica serovar Typhi isolates were resistant to nalidixic acid and 58% (153/263) were multidrug resistant.

Conclusions

Both antibiotics showed an excellent efficacy and safety profile. Both gatifloxacin and azithromycin can be recommended for the treatment of typhoid fever particularly in regions with high rates of multidrug and nalidixic acid resistance. The cost of a 7-day treatment course of gatifloxacin is approximately one third of the cost of azithromycin in Vietnam.

Trial Registration

Controlled-Trials.com ISRCTN67946944     

Two Erysiphe species associated with recent outbreak of soybean powdery mildew: results of molecular phylogenetic analysis based on nuclear rDNA sequences

 Serious outbreaks of powdery mildew by a fungus belonging to the mitosporic genus Oidium subgenus Pseudoidium have been reported on soybean (Glycine max) in a wide area of eastern Asia since 1998. The taxonomic and phylogenetic placement of the causal fungus has not yet been determined because of lack of the perfect stage. We found ascomata having mycelioid appendages on a single leaf of soybean infested by powdery mildew. Molecular phylogenetic analysis was conducted based on a total of 14 sequences of the rDNA internal transcribed spacer (ITS) region from 13 soybean and wild soybean (Glycine soja) materials collected in Japan, Korea, Vietnam, and the United States, combined with 47 sequence data obtained from the DNA databases. It was revealed that two Erysiphe species were associated with the outbreak of soybean powdery mildew. There was 16% difference between the two species in genetic divergence of the ITS sequence. One species with perfect stage has an ITS sequence identical to that of Erysiphe glycines on Amphicarpaea and is identified as Erysiphe glycines based on the ITS sequence and morphology of ascomata. The second species, without the perfect stage, is likely to be Erysiphe diffusa (= Microsphaera diffusa), known as the fungus causing soybean powdery mildew in the United States, because the ITS sequences are identical to those from materials collected in the United States. However, we need materials having ascomata of E. diffusa to confirm the species name. Received: March 15, 2002 / Accepted: May 22, 2002     

Binding of netropsin and 4,6-diamidino-2-phenylindole to an A2T2 DNA hairpin: a comparison of biophysical techniques

Isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and biosensor-surface plasmon resonance (SPR) are evaluated for their accuracy in determining equilibrium constants, ease of use, and range of application. Systems chosen for comparison of the three techniques were the formation of complexes between two minor groove binding compounds, netropsin and 4,6-diamidino-2-phenylindole (DAPI), and a DNA hairpin having the sequence 5'-d(CGAATTCGTCTCCGAATTCG)-3'. These systems were chosen for their structural differences, simplicity (1:1 binding), and binding affinity in the range of interest (K approximately 10(8) M(-1)). The binding affinities determined from all three techniques were in excellent agreement; for example, netropsin/DNA formation constants were determined to be K = 1.7x10(8) M(-1) (ITC), K = 2.4x10(8) M(-1) (DSC), and K = 2.9x10(8) M(-1) (SPR). DSC and SPR techniques have an advantage over ITC in studies of ligands that bind with affinities greater than 10(8) M(-1). The ITC technique has the advantage of determining a full set of thermodynamic parameters, including deltaH, TdeltaS, and deltaC(p) in addition to deltaG (or K). The ITC data revealed complex binding behavior in these minor groove binding systems not detected in the other methods. All three techniques provide accurate estimates of binding affinity, and each has unique benefits for drug binding studies.