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.     

Nonspecific cleavage of proteins using graphene oxide

In this article, we report the intrinsic catalytic activity of graphene oxide (GO) for the nonspecific cleavage of proteins. We used bovine serum albumin (BSA) and a recombinant esterase (rEstKp) from the cold-adapted bacterium Pseudomonas mandelii as test proteins. Cleavage of BSA and rEstKp was nonspecific regarding amino acid sequence, but it exhibited dependence on temperature, time, and the amount of GO. However, cleavage of the proteins did not result in complete hydrolysis into their constituent amino acids. GO also invoked hydrolysis of p-nitrophenyl esters at moderate temperatures lower than those required for peptide hydrolysis regardless of chain length of the fatty acyl esters. Based on the results, the functional groups of GO, including alcohols, phenols, and carboxylates, can be considered as crucial roles in the GO-mediated hydrolysis of peptides and esters via general acid–base catalysis. Our findings provide novel insights into the role of GO as a carbocatalyst with nonspecific endopeptidase activity in biochemical reactions.     

Ligands for FKBP12 Increase Ca2+ Influx and Protein Synthesis to Improve Skeletal Muscle Function

Rapamycin at high doses (2–10 mg/kg body weight) inhibits mammalian target of rapamycin complex 1 (mTORC1) and protein synthesis in mice. In contrast, low doses of rapamycin (10 μg/kg) increase mTORC1 activity and protein synthesis in skeletal muscle. Similar changes are found with SLF (synthetic ligand for FKBP12, which does not inhibit mTORC1) and in mice with a skeletal muscle-specific FKBP12 deficiency. These interventions also increase Ca²⁺ influx to enhance refilling of sarcoplasmic reticulum Ca²⁺ stores, slow muscle fatigue, and increase running endurance without negatively impacting cardiac function. FKBP12 deficiency or longer treatments with low dose rapamycin or SLF increase the percentage of type I fibers, further adding to fatigue resistance. We demonstrate that FKBP12 and its ligands impact multiple aspects of muscle function.     

Experimental evidence for a 9-binding subsite of Bacillus licheniformis thermostable α-amylase

The action pattern of Bacillus licheniformis thermostable α-amylase (BLA) was analyzed using a series of ¹⁴C-labeled and non-labeled maltooligosaccharides from maltose (G2) to maltododecaose (G12). Maltononaose (G9) was the preferred substrate, and yielded the smallest K_m = 0.36 mM, the highest k_cat = 12.86 s⁻¹, and a k_cat/K_m value of 35.72 s⁻¹ mM⁻¹, producing maltotriose (G3) and maltohexaose (G6) as the major product pair. Maltooctaose (G8) was hydrolyzed into two pairs of products: G3 and maltopentaose (G5), and G2 and G6 with cleavage frequencies of 0.45 and 0.30, respectively. Therefore, we propose a model with nine subsites: six in the terminal non-reducing end-binding site and three at the reducing end-binding site in the binding region of BLA.     

Biochemical Characterization of Mutants in Chaperonin Proteins CCT4 and CCT5 Associated with Hereditary Sensory Neuropathy

Hereditary sensory neuropathies are a class of disorders marked by degeneration of the nerve fibers in the sensory periphery neurons. Recently, two mutations were identified in the subunits of the eukaryotic cytosolic chaperonin TRiC, a protein machine responsible for folding actin and tubulin in the cell. C450Y CCT4 was identified in a stock of Sprague-Dawley rats, whereas H147R CCT5 was found in a human Moroccan family. As with many genetically identified mutations associated with neuropathies, the underlying molecular basis of the mutants was not defined. We investigated the biochemical properties of these mutants using an expression system in Escherichia coli that produces homo-oligomeric rings of CCT4 and CCT5. Full-length versions of both mutant protein chains were expressed in E. coli at levels approaching that of the WT chains. Sucrose gradient centrifugation revealed chaperonin-sized complexes of both WT and mutant chaperonins, but with reduced recovery of C450Y CCT4 soluble subunits. Electron microscopy of negatively stained samples of C450Y CCT4 revealed few ring-shaped species, whereas WT CCT4, H147R CCT5, and WT CCT5 revealed similar ring structures. CCT5 complexes were assayed for their ability to suppress aggregation of and refold the model substrate γd-crystallin, suppress aggregation of mutant huntingtin, and refold the physiological substrate β-actin in vitro. H147R CCT5 was not as efficient in chaperoning these substrates as WT CCT5. The subtle effects of these mutations are consistent with the homozygous disease phenotype, in which most functions are carried out during development and adulthood, but some selective function is lost or reduced.