Secondary Signaling Molecules Research Articles

Balanced Duality: H2O2-Based Therapy in Cancer and Its Protective Effects on Non-Malignant Tissues.

Conventional cancer therapy strategies, although centered around killing tumor cells, often lead to severe side effects on surrounding normal tissues, thus compromising the chronic quality of life in cancer survivors. Hydrogen peroxide (H2O2) is a secondary signaling molecule that has an array of functions in both tumor and normal cells, including the promotion of cell survival pathways and immune cell modulation in the tumor microenvironment. H2O2 is a reactive oxygen species (ROS) crucial in cellular homeostasis and signaling (at concentrations maintained under nM levels), with increased steady-state levels in tumors relative to their normal tissue counterparts. Increased steady-state levels of H2O2 in tumor cells, make them vulnerable to oxidative stress and ultimately, cell death. Recently, H2O2-producing therapies-namely, pharmacological ascorbate and superoxide dismutase mimetics-have emerged as compelling complementary treatment strategies in cancer. Both pharmacological ascorbate and superoxide dismutase mimetics can generate excess H2O2 to overwhelm the impaired H2O2 removal capacity of cancer cells. This review presents an overview of H2O2 metabolism in the physiological and malignant states, in addition to discussing the anti-tumor and normal tissue-sparing mechanism(s) of, and clinical evidence for, two H2O2-based therapies, pharmacological ascorbate and superoxide dismutase mimetics.

Ratiometric electrochemical immunoassay based on 2D Co/Fe MOF decorated with toluidine blue and Fc-labeled Schiff base for accurate assay of alpha-fetoprotein in clinical serum

The high level of alpha-fetoprotein (AFP) expression is closely related to hepatocellular carcinoma (HCC). Herein, a dual signal ratiometric electrochemical immunosensor based on chitosan-ferrocenecarboxaldehyde-spindle gold (Chit-Fc-SAu) and Co/Fe metal-organic framework-toluidine blue/polydopamine (Co/Fe MOF-TB/PDA) was proposed for quantitative analysis of AFP. Specifically, Chit-Fc-SAu worked as a substrate to trap more primary antibodies (Ab1) generating the first electrochemical signal from Fc. Thanks to the large specific surface area, the synergistic and electronic effects of Co/Fe MOF nanosheets, and the rich functional groups of PDA, Co/Fe MOF-TB/PDA could load more secondary antibodies (Ab2) and signal molecules (TB) providing another amplified electrochemical signal. In the presence of AFP, Ab1-AFP-Ab2 formed a sandwich structure, and as the AFP concentration increased, the peak current ratio of TB to Fc (ITB/IFc) also increased. The dual signal ratiometric strategy can avoid environmental signal interference and achieve signal self-calibration, thereby improving the accuracy and reproducibility of detection. After a series of exploration, this self-calibrated ratiometric immunosensor exhibited a wide linear range (0.001−200 ng mL−1), a low detection limit (0.34 pg mL−1), and good repeatability. When applied to the assay of clinical serum samples, the detection results of ratiometric sensor were consistent with that of commercial electrochemiluminescence (ECL) immunoassay, significantly superior to that of non-ratiometric sensor. The self-calibrated strategy based on ratiometric sensor helps to improve the accuracy of AFP in clinical diagnosis.

PlzD modifies Vibrio vulnificus foraging behavior and virulence in response to elevated c-di-GMP.

Many free-swimming bacteria propel themselves through liquid using rotary flagella, and mounting evidence suggests that the inhibition of flagellar rotation initiates biofilm formation, a sessile lifestyle that is a nearly universal surface colonization paradigm in bacteria. In general, motility and biofilm formation are inversely regulated by the intracellular second messenger bis-(3´-5´)-cyclic dimeric guanosine monophosphate (c-di-GMP). Here, we identify a protein, PlzD, bearing a conserved c-di-GMP binding PilZ domain that localizes to the flagellar pole in a c-di-GMP-dependent manner and alters the foraging behavior, biofilm, and virulence characteristics of the opportunistic human pathogen, Vibrio vulnificus. Our data suggest that PlzD interacts with components of the flagellar stator to decrease bacterial swimming speed and changes in swimming direction, and these activities are enhanced when cellular c-di-GMP levels are elevated. These results reveal a physical link between a second messenger (c-di-GMP) and an effector (PlzD) that promotes transition from a motile to a sessile state in V. vulnificus.

The Role of PIK3R1 in Metabolic Function and Insulin Sensitivity.

PIK3R1 (also known as p85α) is a regulatory subunit of phosphoinositide 3-kinases (PI3Ks). PI3K, a heterodimer of a regulatory subunit and a catalytic subunit, phosphorylates phosphatidylinositol into secondary signaling molecules involved in regulating metabolic homeostasis. PI3K converts phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3), which recruits protein kinase AKT to the inner leaflet of the cell membrane to be activated and to participate in various metabolic functions. PIK3R1 stabilizes and inhibits p110 catalytic activity and serves as an adaptor to interact with insulin receptor substrate (IRS) proteins and growth factor receptors. Thus, mutations in PIK3R1 or altered expression of PIK3R1 could modulate the activity of PI3K and result in significant metabolic outcomes. Interestingly, recent studies also found PI3K-independent functions of PIK3R1. Overall, in this article, we will provide an updated review of the metabolic functions of PIK3R1 that includes studies of PIK3R1 in various metabolic tissues using animal models, the mechanisms modulating PIK3R1 activity, and studies on the mutations of human PIK3R1 gene.

Dual GGDEF/EAL-Domain Protein RmcA Controls the Type III Secretion System of Pseudomonas aeruginosa by Interaction with CbrB.

Cyclic diguanylate (c-di-GMP) is a major bacterial secondary signaling molecule that controls a multitude of cellular processes. More than 40 genes encoding diguanylate cyclases and phosphodiesterases have been identified in Pseudomonas aeruginosa, and many of them have been intensively investigated. However, the mechanism through which they achieve signaling specificity remains unclear. Here, we revealed that the absence of the dual GGDEF/EAL-domain protein RmcA significantly affected biofilm formation of P. aeruginosa PAO1 and led to upregulated expression of the type III secretion system (T3SS) genes; overexpression of RmcA strongly reduced the expression of T3SS. Further investigation showed that the regulatory function of RmcA was independent of the Gac/Rsm pathway. To identify the interaction partners of RmcA involved in this process, bacterial two-hybrid library screening was performed. We found that RmcA directly interacts with a two-component response regulator CbrB, which is involved in the regulation of biofilm formation and T3SS expression by RmcA. These findings reveal that the dual-domain GGDEF/EAL protein RmcA could achieve specificity of action through physical interaction with CbrB, which extends understanding the complex regulatory network of the c-di-GMP signaling.

The biofilm matrix: multitasking in a shared space.

The biofilm matrix can be considered to be a shared space for the encased microbial cells, comprising a wide variety of extracellular polymeric substances (EPS), such as polysaccharides, proteins, amyloids, lipids and extracellular DNA (eDNA), as well as membrane vesicles and humic-like microbially derived refractory substances. EPS are dynamic in space and time and their components interact in complex ways, fulfilling various functions: to stabilize the matrix, acquire nutrients, retain and protect eDNA or exoenzymes, or offer sorption sites for ions and hydrophobic substances. The retention of exoenzymes effectively renders the biofilm matrix an external digestion system influencing the global turnover of biopolymers, considering the ubiquitous relevance of biofilms. Physico-chemical and biological interactions and environmental conditions enable biofilm systems to morph into films, microcolonies and macrocolonies, films, ridges, ripples, columns, pellicles, bubbles, mushrooms and suspended aggregates - in response to the very diverse conditions confronting a particular biofilm community. Assembly and dynamics of the matrix are mostly coordinated by secondary messengers, signalling molecules or small RNAs, in both medically relevant and environmental biofilms. Fully deciphering how bacteria provide structure to the matrix, and thus facilitate and benefit from extracellular reactions, remains the challenge for future biofilm research.

Curcumin and its Anti-Cancer Properties: Kitchen to the Laboratory

Curcumin is a naturally derived hydrophobic polyphenolic compound extracted from the rhizome (turmeric) of the herb Curcumalonga. It has been used as a common Indian spice in the form of turmeric since time immemorial. Curcumin is an active ingredient ofturmeric and has been found to have many health benefits.Our main aim is to highlight curcumin's biological activity and anti-cancer properties.Curcumin is a bright yellow-colored substance; curcumin plays a vital role in anti-cancer therapy by suppressing cell proliferationand metastasis. Curcumin has regulatory effects on secondary signaling molecules and the further downstream process leading toregulation of transcription factors like nuclear factor-kappa light chain enhancer of activated B cell or NF-kB. Cancer cells have enhancedtelomerase activity; therefore, their DNA never shortens due to the loss of telomeres. Curcumin initiates p53 protein-dependent andprotein-independent G2/M phase cell cycle arrest. Curcumin has plenty of targets like growth factor receptors (Epidermal GrowthFactor Receptor or EGFR), enzymes, and cytokines. Curcumin has poor bioavailability due to its low solubility in water (0.0004mg/mL atnormal pH 7) and hence produces poor effects when administered orally. Curcumin also produces toxicity in patients when consumedin high concentrations.The 21st century is the age of technology. We see that nanotechnology has enabled us to design curcumin-encapsulated, which reducesits bulk size and brings it within 100 nm. Particles like liposomes (phospholipid vesicles), polymer micelles, SLNs, and microspheresare some drug target delivery systems that can solve problems of transporting curcumin. Nanocurcumins can also be extracted frompure curcumin without any carriers. It is under clinical trial where curcumin has been used as an adjuvant with other chemical agentsto control the spread of cancer.

Biotechnological production of cyclic dinucleotides-Challenges and opportunities.

Cyclic dinucleotides (CDNs) are widely used secondary signaling molecules in prokaryotic and eukaryotic cells. As strong agonists of the stimulator of interferon genes, they are of great interest for pharmaceutical applications. In particular, cyclic-GMP-AMP and related synthetic CDNs are promising candidates in preclinical work and even some in clinical phase 1 and 2 studies. The comparison of chemical and biocatalytic synthesis routes elucidated that biological CDN synthesis offers some advantages, such as shorter synthesis time, avoiding complex protective group chemistry, and the access to a new spectrum of CDNs. However, the synthesis of CDNs in preparative quantities is still a challenge, since the chemical synthesis of CDNs suffers from low yields and complex synthetic routes and the enzymatically catalyzed synthesis is limited by low product titers and process stability. We aim to review the latest discoveries and recent trends in chemical and biocatalytic synthesis of CDNs with a focus on the synthesis of a huge variety of CDN derivatives. We furthermore consider the most promising biotechnological processes for CDN production by evaluating key figures of the currently known processes.

Methylglyoxal induces stress signaling and promotes the germination of maize at low temperature.

Maize is sensitive to cold injury, especially during germination. Since cold causes oxidative stress, compounds that promote the accumulation of free radical forms, such as the reactive aldehyde (RA) methylglyoxal (MG), may be suitable to trigger a systemic defense response. In this study, maize seeds were soaked in MG solution for one night at room temperature, before germination test at 13°C. The exogenous MG enhanced the germination and photosynthetic performance of maize at low temperature. Transcriptome analysis, hormonal, and flavonoid profiling indicated MG-induced changes in photosystem antenna proteins, pigments, late embryogenesis abundant proteins, abscisic acid (ABA) derivatives, chaperons, and certain dihydroflavonols, members of the phenylpropanoid pathway. MG-response of the two maize cultivars (A654 and Cm174) were somewhat different, but we recorded higher endogenous hydrogen peroxide (H2 O2 ) and lower nitric oxide (NO) level in at least one of the treated genotypes. These secondary signal molecules may provoke some of the changes in the hormonal, metabolic and gene expression profile. Decreased auxin transport, but increased ABA degradation and cytokinin and jasmonic acid (JA) synthesis, as well as an altered carbohydrate metabolism and transport (amylases, invertases, and SWEET transporters) could have promoted germination of MG-pretreated seeds. While LEA accumulation could have protected against osmotic stress and catalase expression and production of many antioxidants, like para-hydroxybenzoic acid (p-HBA) and anthocyanins may have balanced the oxidative environment for maize germination. Our results showed that MG-pretreatment could be an effective way to promote cold germination and its effect was more pronounced in the originally cold-sensitive maize genotype.

The Nax (SCN7A) channel: an atypical regulator of tissue homeostasis and disease.

Within an articulately characterized family of ion channels, the voltage-gated sodium channels, exists a black sheep, SCN7A (Nax). Nax, in contrast to members of its molecular family, has lost its voltage-gated character and instead rapidly evolved a new function as a concentration-dependent sensor of extracellular sodium ions and subsequent signal transducer. As it deviates fundamentally in function from the rest of its family, and since the bulk of the impressive body of literature elucidating the pathology and biochemistry of voltage-gated sodium channels has been performed in nervous tissue, reports of Nax expression and function have been sparse. Here, we investigate available reports surrounding expression and potential roles for Nax activity outside of nervous tissue. With these studies as justification, we propose that Nax likely acts as an early sensor that detects loss of tissue homeostasis through the pathological accumulation of extracellular sodium and/or through endothelin signaling. Sensation of homeostatic aberration via Nax then proceeds to induce pathological tissue phenotypes via promotion of pro-inflammatory and pro-fibrotic responses, induced through direct regulation of gene expression or through the generation of secondary signaling molecules, such as lactate, that can operate in an autocrine or paracrine fashion. We hope that our synthesis of much of the literature investigating this understudied protein will inspire more research into Nax not simply as a biochemical oddity, but also as a potential pathophysiological regulator and therapeutic target.

Diffracted X-ray blinking measurements of interleukin 15 receptors in the inner/outer membrane of living NK cells

Interleukin 15 receptor (IL-15R) is a transmembrane signalling protein consisting of 3 subsets: α, β (IL-15Rβ), and γ (γc). IL-2 and IL-15 share the signalling domains IL-15Rβ and γc, although they bind to intrinsic α-subsets and non-signalling domains. Additionally, IL-2 and IL-15 play different roles; therefore, there have been many observations of the dynamic behaviours of IL-15R, which are linked to physiological functions.For more practical discrimination between IL-2 and IL-15, a study was designed and carried out in which α-subsets were removed and a cytoplasmic inhibitor was applied to create a simplified environment in which secondary signalling molecules were reduced. We also applied a new measurement method, diffracted X-ray blinking (DXB), to achieve higher accuracy (<0.01 Å).The dynamics of IL-2 binding (confined motion, max range = 0.71 Å) and IL-15 binding (normal motion) in live natural killer cells were different. We also confirmed.that DXB was a suitable method to quantitatively evaluate the transmembrane protein dynamics of inner/outer live cell membranes by labeling the extracellular domain since the measurements were dependent on the cytosolic environment.

Bacterial second messenger 3\u2032,5\u2032-cyclic diguanylate attracts Caenorhabditis elegans and suppresses its immunity

Cyclic di-nucleotides are important secondary signaling molecules in bacteria that regulate a wide range of processes. In this study, we found that Caenorhabditis elegans can detect and are attracted to multiple signal molecules produced by Vibrio cholerae, specifically the 3′,5′-cyclic diguanylate (c-di-GMP), even though this bacterium kills the host at a high rate. C-di-GMP is sensed through C. elegans olfactory AWC neurons, which then evokes a series of signal transduction pathways that lead to reduced activity of two key stress response transcription factors, SKN-1 and HSF-1, and weakened innate immunity. Taken together, our study elucidates the role of c-di-GMP in interkingdom communication. For C. elegans, bacterial c-di-GMP may serve as a cue that they can use to detect food. On the other hand, preexposure to low concentrations of c-di-GMP may impair their immune response, which could facilitate bacterial invasion and survival.

Diguanylate Cyclases in Vibrio cholerae: Essential Regulators of Lifestyle Switching

Biofilm formation in Vibrio cholerae empowers the bacteria to lead a dual lifestyle and enhances its infectivity. While the formation and dispersal of the biofilm involves multiple components—both proteinaceous and non-proteinaceous, the key to the regulatory control lies with the ubiquitous secondary signaling molecule, cyclic-di-GMP (c-di-GMP). A number of different cellular components may interact with c-di-GMP, but the onus of synthesis of this molecule lies with a class of enzymes known as diguanylate cyclases (DGCs). DGC activity is generally associated with proteins possessing a GGDEF domain, ubiquitously present across all bacterial systems. V. cholerae is also endowed with multiple DGCs and information about some of them have been pouring in over the past decade. This review summarizes the DGCs confirmed till date in V. cholerae, and emphasizes the importance of DGCs and their product, c-di-GMP in the virulence and lifecycle of the bacteria.

Abstract PR10: Disruption of IL6-mediated paracrine signaling to prevent pulmonary metastasis

Abstract Outcomes for patients diagnosed with osteosarcoma (OS) hinge primarily on whether or not they develop pulmonary metastasis. Looking to identify novel opportunities for therapeutic intervention, we have sought mechanisms that facilitate colonization of lung tissues by osteosarcoma cells. We have shown that metastatic colonization of lung by OS cells requires their IL6 and CXCL8 production, which is elicited upon the interaction of OS cells with lung tissues. The lung-derived signals that trigger this behavior and the effector mechanisms by which IL6 and CXCL8 facilitate metastatic colonization remain unknown. Here, we hypothesized that the interaction of OS cells lung epithelium triggers production of cytokines that establish paracrine signal amplification loops. We postulated that OS cell-derived IL6 and CXCL8 stimulate lung-resident cells to produce secondary signaling molecules that are, in turn, capable of stimulating OS cells. If our paracrine loop hypothesis is correct, we would expect that these lung cell-derived factors elicit further production of IL6 and CXCL8, resulting in a feed-forward paracrine signaling. Our lab has shown that soluble factors produced by lung epithelium profoundly increase IL6 and CXCL8 production by OS cells. In order to identify the specific factors responsible for this effect, we used RNA-seq to identify soluble ligands produced by lung epithelial cells that could activate cognate receptors produced by OS cells. Epithelial cell production of a subset of these soluble ligands increased when cells were exposed to supernatants from OS cells. We then performed a similar analysis using single-cell RNA-seq in mouse lungs bearing metastatic OS tumors, which identified many of the same ligands and receptors. Eleven candidate epithelial-to-tumor ligand-receptor interactions were identified in both the in vitro and the in vivo analysis. Interpreting these results in the context of prior known biology, one of these factors, IL-1, stood out as a prime candidate for a lung-to-tumor signal that would elicit IL6 and CXCL8 production in disseminated OS cells. We have since shown that IL-1 is upregulated in lung cells upon interaction with OS cells, that the cognate receptor IL1R1 is increased on OS cells following exposure to lung cell conditioned media, and that stimulation of OS cells with IL-1β drastically increases production of both IL6 and CXCL8. We have also verified production of IL-1α/β in lung epithelial cells and are working to confirm these findings in additional human-derived lung cell models. Our results suggest that this lung cell-tumor cell paracrine loop may play a mechanistic role in the colonization of lung tissue by OS. If true, the therapeutic effects of inhibiting this imminently targetable pathway on OS lung metastasis should be explored. Such studies are currently under way. This abstract is also being presented as Poster A61. Citation Format: John Hinckley, Amy Gross, Ryan Roberts. Disruption of IL6-mediated paracrine signaling to prevent pulmonary metastasis [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr PR10.

Improving Photosynthetic Capacity, Alleviating Photosynthetic Inhibition and Oxidative Stress Under Low Temperature Stress With Exogenous Hydrogen Sulfide in Blueberry Seedlings.

In this study, we investigated the mechanism of photosynthesis and physiological function of blueberry leaves under low temperature stress (4–6°C) by exogenous hydrogen sulfide (H2S) by spraying leaves with 0.5 mmol·L–1 NaHS (H2S donor) and 200 μmol·L–1 hypotaurine (Hypotaurine, H2S scavenger). The results showed that chlorophyll and carotenoid content in blueberry leaves decreased under low temperature stress, and the photochemical activities of photosystem II (PSII) and photosystem I (PSI) were also inhibited. Low temperature stress can reduce photosynthetic carbon assimilation capacity by inhibiting stomatal conductance (Gs) of blueberry leaves, and non-stomatal factors also play a limiting role at the 5th day of low temperature stress. Low temperature stress leads to the accumulation of Pro and H2O2 in blueberry leaves and increases membrane peroxidation. Spraying leaves with NaHS, a donor of exogenous H2S, could alleviate the degradation of chlorophyll and carotenoids in blueberry leaves caused by low temperature and reduce the photoinhibition of PSII and PSI. The main reason for the enhancement of photochemical activity of PSII was that exogenous H2S promoted the electron transfer from QA to QB on PSII acceptor side under low temperature stress. In addition, it promoted the accumulation of osmotic regulator proline under low temperature stress and significantly alleviated membrane peroxidation. H2S scavengers (Hypotaurine) aggravated photoinhibition and the degree of oxidative damage under low temperature stress. Improving photosynthetic capacity as well as alleviating photosynthetic inhibition and oxidative stress with exogenous H2S is possible in blueberry seedlings under low temperature stress.

Paracrine signaling in islet function and survival.

The pancreatic islet is a dense cellular network comprised of several cell types with endocrine function vital in the control of glucose homeostasis, metabolism, and feeding behavior. Within the islet, endocrine hormones also form an intricate paracrine network with supportive cells (endothelial, neuronal, immune) and secondary signaling molecules regulating cellular function and survival. Modulation of these signals has potential consequences for diabetes development, progression, and therapeutic intervention. Beta cell loss, reduced endogenous insulin secretion, and dysregulated glucagon secretion are hallmark features of both type 1 and 2 diabetes that not only impact systemic regulation of glucose, but also contribute to the function and survival of cells within the islet. Advancing research and technology have revealed new islet biology (cellular identity and transcriptomes) and identified previously unrecognized paracrine signals and mechanisms (somatostatin and ghrelin paracrine actions), while shifting prior views of intraislet communication. This review will summarize the paracrine signals regulating islet endocrine function and survival, the disruption and dysfunction that occur in diabetes, and potential therapeutic targets to preserve beta cell mass and function.

Solution Structures of a G-Quadruplex Bound to Linear- and Cyclic-Dinucleotides.

Cyclic dinucleotides have emerged as important secondary messengers and cell signaling molecules that regulate several cell responses. A guanine-deficit G-quadruplex structure formation by a sequence containing (4n - 1) guanines, n denoting the number of G-tetrad layers, was previously reported. Here, a (4n - 1) G-quadruplex structure is shown to be capable of binding guanine-containing dinucleotides in micromolar affinity. The guanine base of the dinucleotides interacts with a vacant G-triad, forming four additional Hoogsteen hydrogen bonds to complete a G-tetrad. Solution structures of two complexes, both comprised of a (4n - 1) G-quadruplex structure, one bound to a linear dinucleotide (d(AG)) and the other to a cyclic dinucleotide (cGAMP), are solved using NMR spectroscopy. The latter suggests sufficiently strong interaction between the guanine base of the dinucleotide and the vacant G-triad, which acts as an anchor point of binding. The binding interfaces from the two solution structures provide useful information for specific ligand design. The results also infer that other guanine-containing metabolites of a similar size have the capability of binding G-quadruplexes, potentially affecting the expression of the metabolites and functionality of the bound G-quadruplexes.

Highly Efficient Preparation of Cyclic Dinucleotides via Engineering of Dinucleotide Cyclases in Escherichia coli.

Cyclic dinucleotides (CDNs) are widely used secondary signaling molecules in bacterial and mammalian cells. The family of CDNs includes c-di-GMP, c-di-AMP and two distinct versions of hybrid cGAMPs. Studies related to these CDNs require large doses that are relatively expensive to generate by current methods. Here we report what to our knowledge is the first feasible microbial-based method to prepare these CDNs including c-di-GMP, 3′3′-cGAMP and 2′3′-cGAMP. The method mainly includes two parts: producing high yield of CDNs by engineering the overexpression of the proper dinucleotide cyclases (DNCs) and other related proteins in Escherichia coli, and purifying the bacteria-produced CDNs by a unified and simple process involving a STING affinity column, macroporous adsorption resin and C18 reverse-phase liquid chromatography. After purification, we obtained the diammonium salts of c-di-GMP, 3′3′-cGAMP and 2′3′-cGAMP with weight purity of >99, >96, >99% and in yields of >68, >26, and >82 milligrams per liter of culture, respectively. This technological platform enables the production of CDNs from cheaper material, provides a sustainable source of CDNs for scientific investigation, and can easily be further developed to prepare CDNs on a large scale for industry.

Induction of signal molecules and expression of functional genes after Pichia pastoris stimulation in Glycyrrhiza uralensis Fisch adventitious roots.

Glycyrrhiza uralensis Fisch is threatened by over-development and consumption, and therefore, in urgent need of protection. Elicitation is considered to be an effective strategy to enhance the secondary metabolites in plant cell and organ cultures. Secondary metabolite, signal molecules, and gene expression in adventitious roots were studied by HPLC-ESI-MSn , commercially available kits and qRT-PCR method, respectively. In the present study, with the addition of linolenic acid, linoleic acid, and Pichia pastoris, the highest concentration of metabolites was achieved by P. pastoris treatment. The contents of total flavonoids (7.16mg/g) and polysaccharide (149.76mg/g) peaked at 100mg/L of P. pastoris, which increased by 3.09-fold and 3.28-fold compared with the control, respectively. However, the highest concentration of glycyrrhizic acid (0.62mg/g) and glycyrrhetinic acid (0.29mg/g) were obtained in 200mg/L of P. pastoris and which were 3.89-fold and 2.42-fold more than the control group, respectively. ESI-MSn analysis indicated that licoricesaponine B2, licoricesapoine G2, licoricesaponine J2, ononin, uralenin, gancaonin C were only identified in the P. pastoris treatment group. Furthermore, P. pastoris also enhanced accumulation of salicylic acid, jasmonic acid, nitric oxide and activities of antioxidant enzymes involved in the plant defense response. In addition, the transcriptional activity of genes involved in glycyrrhizic acid biosynthesis was significantly increased under the treatment of P. pastoris. The results provided a scientific evidence for the further exploitation of G. uralensis adventitious roots and clinical medication. PRACTICAL APPLICATIONS: This study provided an effective strategy to enhance metabolites by Pichia pastoris treatment in adventitious roots of G. uralensis. The data provide a scientific evidence for the further exploitation of G. uralensis adventitious roots and clinical medication.

Cucumber Phospholipase D alpha gene overexpression in tobacco enhanced drought stress tolerance by regulating stomatal closure and lipid peroxidation

BackgroundPlant phospholipase D (PLD), which can hydrolyze membrane phospholipids to produce phosphatidic acid (PA), a secondary signaling molecule, has been proposed to function in diverse plant stress responses. Both PLD and PA play key roles in plant growth, development, and cellular processes. PLD was suggested to mediate the regulation of stomatal movements by abscisic acid (ABA) as a response to water deficit. In this research, we characterized the roles of the cucumber phospholipase D alpha gene (CsPLDα, GenBank accession number EF363796) in the growth and tolerance of transgenic tobacco (Nicotiana tabacum) to drought stress.ResultsThe CsPLDα overexpression in tobacco lines correlated with the ABA synthesis and metabolism, regulated the rapid stomatal closure in drought stress, and reduced the water loss. The NtNCED1 expression levels in the transgenic lines and wild type (WT) were sharply up-regulated after 16 days of drought stress compared with those before treatment, and the expression level in the transgenic lines was significantly higher than that in the WT. The NtAOG expression level evidently improved after 8 and 16 days compared with that at 0 day of treatment and was significantly lower in the transgenic lines than in the WT. The ABA content in the transgenic lines was significantly higher than that in the WT. The CsPLDα overexpression could increase the osmolyte content and reduce the ion leakage. The proline, soluble sugar, and soluble protein contents significantly increased. By contrast, the electrolytic leakage and malondialdehyde accumulation in leaves significantly decreased. The shoot and root fresh and dry weights of the overexpression lines significantly increased. These results indicated that a significant correlation between CsPLDα overexpression and improved resistance to water deficit.ConclusionsThe plants with overexpressed CsPLDα exhibited lower water loss, higher leaf relative water content, and heavier fresh and dry matter accumulation than the WT. We proposed that CsPLDα was involved in the ABA-dependent pathway in mediating the stomatal closure and preventing the elevation of intracellular solute potential.