Untying arsenite tolerance mechanisms in contrasting maize genotypes attributed to NIPs-mediated controlled influx and root-to-shoot translocation, redox homeostasis and phytochelatin-mediated detoxification pathway.

Contamination of ground water and soil with toxic metalloids like arsenic (As) poses a serious hazard to the global agricultural food production. One of the best ways to restrict entry of As into the food chain is selection of germplasms which accrue extremely low level of As in grains. Here, we screened diverse maize genotypes under high arsenite (100 µM AsIII) stress and identified PMI-PV-9 and PMI-PV-3 as AsIII-tolerant and -sensitive maize genotype respectively. Expression of genes associated with As uptake, vacuolar sequestration, biosynthesis of phytochelatins, root-to-shoot translocation, in vivo ROS generation, fine tuning of antioxidant defense system, DNA and membrane damage, H2O2 and superoxide anion (O2•-) levels were compared among the selected genotypes. PMI-PV-9 plants performed much better than PMI-PV-3 in terms of plant growth with no visible symptom of As toxicity. Susceptibility of PMI-PV-3 to AsIII stress may be attributed to comparatively low expression of genes involved in phytochelatins (PCs) biosynthesis. Concomitant decrease in ABCC1 expression might be another key factor for futile sequestration of AsIII into root vacuoles. Moreover, up-regulation of ZmNIP3;1 might contribute in high root-to-leaf As translocation. Substantial spike in H2O2, O2•- and MDA levels indicates that PMI-PV-3 plants have experienced more oxidative stress than PMI-PV-9 plants. Appearance of prominent deep brown and dark blue spots/stripes on leaves as revealed after DAB and NBT staining respectively suggest severe oxidative burst in PMI-PV-3 plants. Marked reduction in DHAR and MDAR activity rendered PMI-PV-3 cells to recycle ascorbate pool ineffectively, which might have exacerbated their susceptibility to AsIII stress. In a nutshell, incompetent PCs mediated detoxification system and disruption of cellular redox homeostasis owing to feeble antioxidant defence system resulting oxidative burst might be the prime reasons behind reduced performance of PMI-PV-3 plants under AsIII stress.

Localization of PPM1H phosphatase tunes Parkinson's disease-linked LRRK2 kinase-mediated Rab GTPase phosphorylation and ciliogenesis.

PPM1H phosphatase reverses Parkinson's disease-associated, Leucine Rich Repeat Kinase 2-mediated Rab GTPase phosphorylation. We show here that PPM1H relies on an N-terminal amphipathic helix for Golgi localization. The amphipathic helix enables PPM1H to bind to liposomes in vitro, and small, highly curved liposomes stimulate PPM1H activity. We artificially anchored PPM1H to the Golgi, mitochondria, or mother centriole. Our data show that regulation of Rab10 GTPase phosphorylation requires PPM1H access to Rab10 at or near the mother centriole. Moreover, poor colocalization of Rab12 explains in part why it is a poor substrate for PPM1H in cells but not in vitro. These data support a model in which localization drives PPM1H substrate selection and centriolar PPM1H is critical for regulation of Rab GTPase-regulated ciliogenesis. Moreover, Golgi localized PPM1H may maintain active Rab GTPases on the Golgi to carry out their nonciliogenesis-related functions in membrane trafficking.

microRNAomic profiling of maize root reveals multifaceted mechanisms to cope with Cr (VI) stress.

Chromium (Cr) contamination of soil and water poses serious threats to agricultural crop production. MicroRNAs (miRNAs) are conserved, non-coding small RNAs that play pivotal roles in plant growth, development and stress responses through fine-tuning of post-transcriptional gene expression. To better understand the molecular circuit of Cr-responsive miRNAs, two sRNA libraries were prepared from control and Cr (VI) [100 ppm] exposed maize roots. Using deep sequencing, we identified 80 known (1 up and 79 down) and 18 downregulated novel miRNAs from Cr (VI) challenged roots. Gene ontology (GO) analysis reveals that predicted target genes of Cr (VI) responsive miRNAs are potentially involved in diverse cellular and biological processes including plant growth and development (miR159c, miR164d, miR319b-3p and zma_25.145), redox homeostasis (miR528-5p, miR396a-5p and zma_9.132), heavy metal uptake and detoxification (miR159f-5p, 164e-5p, miR408a, miR444f and zma_2.127), signal transduction (miR159f, miR160a-5p, miR393a-5p, miR408-5p and zma_43.158), cell signalling (miR156j, 159c-5p, miR166c-5p and miR398b). Higher accumulation of Cr in maize roots might be due to upregulation of ABC transporter G family member 29 targeted by miR444f. Instead of isolated increase in SOD expression, significant decline in GSH:GSSH ratio and histochemical staining strongly suggest Cr (VI) stress mediated disruption of ROS scavenging machinery thus unbalancing normal cellular homeostasis. Moreover, miR159c-mediated enhanced expression of GAMYB might be a reason for impaired root growth under Cr (VI) stress. In a nutshell, the present microRNAomic study sheds light on the miRNA-target gene regulatory network involved in adaptive responses of maize seedlings to Cr (VI) stress.

Large area multi-filamentary plasma source for large volume plasma device-upgrade.

This paper discusses the salient features and plasma performance of the newly installed Large Area Multi-Filamentary Plasma Source (LAMPS) in large volume plasma device-upgrade. The plasma source is designed to exhibit a plasma electron density of ∼1018 m-3, low electron temperature (∼eV), and a uniform plasma cross section of 2.54 m2. The directly heated LAMPS emits accelerated primary energetic electrons when it is biased with a negative discharge voltage with respect to the anode. The hairpin shaped tungsten (W) filaments, each of diameter 0.5 mm and length 180 mm, are heated to a temperature of 2700 K by feeding ∼19.5A to each filament. The LAMPS consists of 162 numbers of filaments, and it has been successfully operated with a total investment of 50 kW of electrical power. The LAMPS as a laboratory plasma source is characterized by large operational life, ease of handling, better compatibility to high pressure conditions, and advantages over other contemporary plasma sources, viz., oxide coated cathodes, RF based sources, and helicon sources, when producing plasma over large cross sections and fill volumes. Pulsed argon plasma is produced with quiescence (δnene≪1%) using LAMPS for the duration of 50 ms and a reasonably good radial uniformity (Ln = 210 cm) is achieved. Good axial uniformity is also observed over the entire length of the device. Initial measurements on plasma parameters have yielded plasma density of ∼2×1017m-3 with existing set of filaments. A plasma density of ∼1018 m-3 is envisaged for larger thickness of filaments, such as 0.75 and 1.0 mm, with the existing plasma source assembly setup.

A feed-forward pathway drives LRRK2 kinase membrane recruitment and activation.

Activating mutations in the leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's disease, and previously we showed that activated LRRK2 phosphorylates a subset of Rab GTPases (Steger et al., 2017). Moreover, Golgi-associated Rab29 can recruit LRRK2 to the surface of the Golgi and activate it there for both auto- and Rab substrate phosphorylation. Here, we define the precise Rab29 binding region of the LRRK2 Armadillo domain between residues 360-450 and show that this domain, termed 'site #1,' can also bind additional LRRK2 substrates, Rab8A and Rab10. Moreover, we identify a distinct, N-terminal, higher-affinity interaction interface between LRRK2 phosphorylated Rab8 and Rab10 termed 'site #2' that can retain LRRK2 on membranes in cells to catalyze multiple, subsequent phosphorylation events. Kinase inhibitor washout experiments demonstrate that rapid recovery of kinase activity in cells depends on the ability of LRRK2 to associate with phosphorylated Rab proteins, and phosphorylated Rab8A stimulates LRRK2 phosphorylation of Rab10 in vitro. Reconstitution of purified LRRK2 recruitment onto planar lipid bilayers decorated with Rab10 protein demonstrates cooperative association of only active LRRK2 with phospho-Rab10-containing membrane surfaces. These experiments reveal a feed-forward pathway that provides spatial control and membrane activation of LRRK2 kinase activity.

Impact of CuO nanoparticles on maize: Comparison with CuO bulk particles with special reference to oxidative stress damages and antioxidant defense status.

The present study aimed to systematically investigate the particle size effects of copper (II) oxide [CuO nanoparticles (<50 nm) and CuO bulk particles (<10 µm)] on maize (Zea mays L.). Bioaccumulation of Cu, in vivo ROS generation, membrane damage, transcriptional modulation of antioxidant genes, cellular redox status of glutathione and ascorbate pool, expression patterns of COPPER TRANSPORTER 4 and stress responsive miRNAs (miR398a, miR171b, miR159f-3p) with their targets were investigated for better understanding of the underlying mechanisms and the extent of CuO nanoparticles and CuO bulk particles induced oxidative stress damages. More restricted seedling growth, comparatively higher membrane injury, marked decline in the levels of chlorophylls and carotenoids and severe oxidative burst were evident in CuO bulk particles challenged leaves. Dihydroethidium and CM-H2DCFDA staining further supported elevated reactive oxygen species generation in CuO bulk particles stressed roots. CuO bulk particles exposed seedlings accumulated much higher amount of Cu in roots as compared to CuO nanoparticles stressed plants with low root-to-shoot Cu translocation. Moderately high GR expression with maintenance of a steady GSH-GSSG ratio in CuO nanoparticles challenged leaves might be accountable for their rather improved performance under stressed condition. miR171b-mediated enhanced expression of SCARECROW 6 might participate in the marked decline of chlorophyll content in CuO bulk particles exposed leaves. Ineffective recycling of AsA pool is another decisive feature of inadequate performance of CuO bulk particles stressed seedlings in combating oxidative stress damages. Taken together, our findings revealed that toxicity of CuO bulk particles was higher than CuO nanoparticles and the adverse effects of CuO bulk particles on maize seedlings might be due to higher Cu ions dissolution.

Development of electronic record-keeping software for remote participation in Large Volume Plasma Device upgrade using Angular 2 and NodeJS web technologies.

In an era of digital transformation and collaborations, the Web Information System (WIS) becomes an essential requirement for the information and data sharing of large experimental facilities among users. With the enhancement in the capabilities and performance of web technologies, sharing of experimental data using a flexible, modular, secure, and robust mechanism is feasible. In this direction, the Large Volume Plasma Device (LVPD), an experimental device dedicated for carrying out investigations for unfolding physical phenomena of relevance to Earth's magnetosphere and fusion plasmas, also adopts web-based electronic record keeping for its operation. The nature of investigations is concerned with plasma turbulence of electron scale, induced anomalous plasma transport and mitigation of energetic electrons by excited whistler turbulence that requires large scale, and simultaneous multiple point measurements from different electrostatic and electro-magnetic diagnostics. This paper discusses the WIS implementation in LVPD for the experimental configuration, information logging, and preliminary data analysis. The architecture of the system is spread over three tiers covering application, data, and presentation layers. The presentation layer is developed using the Angular 2 framework on the progressive web application architecture. The application and data layers are developed using NodeJS and PostgreSQL, respectively. The novelty of this paper lies in the integrated application development approach and applicability of the latest web technologies in the scientific and experimental domains. This paper discusses the literature survey of similar developments at other places, requirements, scopes, development artifacts, adapted tools and technologies, obtained results from actual plasma discharges of LVPD, and future enhancements.

Regulation of Leishmania major PAS domain-containing phosphoglycerate kinase by cofactor Mg2+ ion at neutral pH.

Recently, we described the PAS domain-containing phosphoglycerate kinase (PGK) from Leishmania major (LmPAS-PGK) that shows acidic pH (5.5)-dependent optimum catalytic activity. The PAS domain of LmPAS-PGK is expected to regulate PGK activity during catalysis, but the mechanism of regulation by PAS domain at the molecular level is uncharacterized. In this work, we have utilized the full-length, PAS domain-deleted, and mutant enzymes to measure the enzymatic activity in the presence of divalent cation at various pH values. Catalytic activity measurement indicates that Mg2+ binding through PAS domain inhibits the PGK activity at pH 7.5, and this inhibition is withdrawn at pH 5.5. To identify the Mg2+ binding residues of the PAS domain, we exploited a systematic mutational analysis of all (four) His residues in the PAS domain for potential divalent cation binding. Replacement of His-57 with alanine resulted in depression in the presence of Mg2+ at pH 7.5, but H71A, H89A, and H111A showed similar characteristics with respect to the wild-type protein. Fluorescence and isothermal titration calorimetry studies revealed that H57 is responsible for Mg2+ binding in the absence of substrates. Thus, the protonated form of His57 at acidic pH 5.5 destabilizes the Mg2+ binding in the PAS domain, which is an essential requirement in the wild-type LmPAS-PGK for a conformational alteration in the sensor domain that, sequentially, activates the PGK domain, resulting in the synthesis of higher amounts of ATP.

Assessment of ZnO-NPs toxicity in maize: An integrative microRNAomic approach.

Rapid expansion of nanotechnology and indiscriminate discharge of metal oxide nanoparticles (NPs) into the environment pose a serious hazard to the ecological receptors including plants. To better understand the role of miRNAs in ZnO-NPs stress adaptation, two small RNA libraries were prepared from control and ZnO-NPs (800 ppm, <50 nm particle size) stressed maize leaves. Meager performance of ZnO-NPs treated seedlings was associated with elevated tissue zinc accumulation, enhanced ROS generation, loss of root cell viability, increased foliar MDA content, decrease in chlorophyll and carotenoids contents. Deep sequencing identified 3 (2 known and 1 novel) up- and 77 (73 known and 4 novel) down-regulated miRNAs from ZnO-NPs challenged leaves. GO analysis reveals that potential targets of ZnO-NPs responsive miRNAs regulate diverse biological processes viz. plant growth and development (miR159f-3p, zma_18), ROS homeostasis (miR156b, miR166l), heavy metal transport and detoxification (miR444a, miR167c-3p), photosynthesis (miR171b) etc. Up-regulation of SCARECROW 6 in ZnO-NPs treated leaves might be responsible for suppression of chlorophyll biosynthesis leading to yellowing of leaves. miR156b.1 mediated up-regulation of CALLOSE SYNTHASE also does not give much protection against ZnO-NPs treatment. Taken together, the findings shed light on the miRNA-guided stress regulatory networks involved in plant adaptive responses to ZnO-NPs stress.

PAS domain-containing phosphoglycerate kinase deficiency in Leishmania major results in increased autophagosome formation and cell death.

Per-Arnt-Sim (PAS) domains are structurally conserved and present in numerous proteins throughout all branches of the phylogenetic tree. Although PAS domain-containing proteins are major players for the adaptation to environmental stimuli in both prokaryotic and eukaryotic organisms, these types of proteins are still uncharacterized in the trypanosomatid parasites, Trypanosome and Leishmania In addition, PAS-containing phosphoglycerate kinase (PGK) protein is uncharacterized in the literature. Here, we report a PAS domain-containing PGK (LmPAS-PGK) in the unicellular pathogen Leishmania The modeled structure of N-terminal of this protein exhibits four antiparallel ß sheets centrally flanked by α helices, which is similar to the characteristic signature of PAS domain. Activity measurements suggest that acidic pH can directly stimulate PGK activity. Localization studies demonstrate that the protein is highly enriched in the glycosome and its presence can also be seen in the lysosome. Gene knockout, overexpression and complement studies suggest that LmPAS-PGK plays a fundamental role in cell survival through autophagy. Furthermore, the knockout cells display a marked decrease in virulence when host macrophage and BALB/c mice were infected with them. Our work begins to clarify how acidic pH-dependent ATP generation by PGK is likely to function in cellular adaptability of Leishmania.

Loss of virulence in NAD(P)H cytochrome b5 oxidoreductase deficient Leishmania major.

Leishmania promastigotes have the ability to synthesize essential polyunsaturated fatty acids de novo and can grow in lipid free media. Recently, we have shown that NAD(P)H cytochrome b5 oxidoreductase (Ncb5or) enzyme in Leishmania acts as the redox partner for Δ12 fatty acid desaturase, which catalyses the conversion of oleate to linoleate. So far, the exact role of Leishmania derived linoleate synthesis is still incomplete in the literature. The viability assay by flow cytometry as well as microscopic studies suggests that linoleate is an absolute requirement for Leishmania promastigote survival in delipidated media. Western blot analysis suggested that infection with log phase linoleate deficient mutant (KO) results in increased level of NF-κBp65, IκB and IKKß phosphorylation in RAW264.7 cells. Similarly, the log phase KO infected RAW264.7 cells show dramatic increment of COX-2 expression and TNF-α secretion, compared to control or Ncb5or complement (CM) cell lines. The activation of inflammatory signaling pathways by KO mutant is significantly reduced when the RAW264.7 cells are pre-treated with BSA bound linoleate. Together, these findings confirmed that the leishmanial linoleate inhibits both COX-2 and TNF-α expression in macrophage via the inactivation of NF-κB signaling pathway. The stationary phase of KO promastigotes shows avirulence after infection in macrophages as well as inoculation into BALB/c mice; whereas CM cell lines show virulence. Collectively, these data provide strong evidence that de novo linoleate synthesis in Leishmania is an essential for parasite survival at extracellular promastigote stage as well as intracellular amastigote stage.

Control of catalysis in globin coupled adenylate cyclase by a globin-B domain.

The globin coupled heme containing adenylate cyclase from Leishmania major (HemAC-Lm) has two globin domains (globin-A and globin-B). Globin-B domain (210-360 amino acids) may guide the interaction between globin-A and adenylate cyclase domains for the regulation of catalysis. We investigated the role of globin-B domain in HemAC-Lm by constructing a series of mutants namely Δ209 (209 amino acids deleted), Δ360 (360 amino acids deleted), H161A, H311A and H311A-Δ209. Spectroscopic data suggest that the Δ209 and H311A-Δ209 proteins to be Fe(2+)-O2 form and apo form, respectively, indicating that His311 residue in the globin-B domain is crucial for heme binding in Δ209 protein. However, the H311A mutant is still of the Fe(2+)-O2 form whereas H161A mutant shows the apo form, indicating that only His161 residue in the globin-A domain is responsible for heme binding in full length enzyme. cAMP measurements suggest that the activities of Δ360 and Δ209 proteins were ∼10 and ∼1000 times lesser than full length enzyme, respectively, leading to the fact that globin-B domain inhibited catalysis rather than activation in absence of globin-A domain. These data suggest that the O2 bound globin-A domain in HemAC-Lm allows the best cooperation of the catalytic domain interactions to generate optimum cAMP.