Nitrogen fertiliser and establishment method affect growth,yield and nitrogen use efficiency of rice under alternate wetting and drying irrigation

Sustainable rice production through selection of best suitable cultivar, water-efficient crop establishment method and optimum nitrogen (N) management practice is needed to feed the growing world population. Two polyhouse experiments were conducted to evaluate the response of rice to different rates and schedules of N application under different establishment methods subjected to alternate wetting and drying (AWD) irrigation. In the first experiment, the response of two rice cultivars (Pathumthani 1, RD57) under three establishment methods (transplanting [TP], wet direct seeding [WDS], dry direct seeding [DDS]) and five N rates (0 [N₀], 30 [N₃₀], 60 [N₆₀], 90 [N₉₀], 120 [N₁₂₀] kg ha⁻¹) was evaluated. The second experiment consisted of the same cultivars and establishment methods, but with four N application schedules (T₁: 100% at basal; T₂: 75% + 25% at basal and at active tillering, respectively; T₃: 50% + 25% + 25% at basal, at active tillering and at panicle initiation, respectively; and T₄: 25% + 25% + 25% + 25% each at basal, at active tillering, at panicle initiation and at early flowering or just before heading starts) applied at the rate of N₆₀ kg ha⁻¹. Plants were maintained under AWD irrigation (soil was saturated by applying water whenever soil water potential drops to −5 kPa during the implementation period) in both experiments. RD57 performed better than Pathumthani 1 having higher shoot dry matter, panicle number, grain yield, total N uptake and apparent N recovery efficiency. TP gave better response than WDS and DDS regardless of cultivars. Application of N₁₂₀ resulted in better growth, yield and its components and total N uptake regardless of establishment methods and cultivars. Increasing N rate decreased N use efficiency (NUE). Scheduling the interval of N application to two or three times (T₂ or T₃) for RD57 and Pathumthani 1, respectively, provided overall better results, and could be recommended for the tested rice cultivars. An optimal N rate and selection of critical growth stages for N application would be very effective for maximising yield and NUE under the water-saving cultivation technique of AWD irrigation.     

Microbial diversity and function in crystalline basement beneath the Deccan Traps explored in a 3 km borehole at Koyna,western India

Deep terrestrial subsurface represents a huge repository of global prokaryotic biomass. Given its vastness and importance, microbial life within the deep subsurface continental crust remains under-represented in global studies. We characterize the microbial communities of deep, extreme and oligotrophic realm hosted by crystalline Archaean granitic rocks underneath the Deccan Traps, through sampling via 3000 m deep scientific borehole at Koyna, India through metagenomics, amplicon sequencing and cultivation-based analyses. Gene sequences 16S rRNA (7.37 × 10⁶) show considerable bacterial diversity and the existence of a core microbiome (5724 operational taxonomic units conserved out of a total 118,064 OTUs) across the depths. Relative abundance of different taxa of core microbiome varies with depth in response to prevailing lithology and geochemistry. Co-occurrence network analysis and cultivation attempt to elucidate close interactions among autotrophic and organotrophic bacteria. Shotgun metagenomics reveals a major role of autotrophic carbon fixation via the Wood–Ljungdahl pathway and genes responsible for energy and carbon metabolism. Deeper analysis suggests the existence of an ‘acetate switch’, coordinating biosynthesis and cellular homeostasis. We conclude that the microbial life in the nutrient- and energy-limited deep granitic crust is constrained by the depth and managed by a few core members via a close interplay between autotrophy and organotrophy.     

Dynamic coupling of residues within proteins as a mechanistic foundation of many enigmatic pathogenic missense variants

Many pathogenic missense mutations are found in protein positions that are neither well-conserved nor fall in any known functional domains. Consequently, we lack any mechanistic underpinning of dysfunction caused by such mutations. We explored the disruption of allosteric dynamic coupling between these positions and the known functional sites as a possible mechanism for pathogenesis. In this study, we present an analysis of 591 pathogenic missense variants in 144 human enzymes that suggests that allosteric dynamic coupling of mutated positions with known active sites is a plausible biophysical mechanism and evidence of their functional importance. We illustrate this mechanism in a case study of β-Glucocerebrosidase (GCase) in which a vast majority of 94 sites harboring Gaucher disease-associated missense variants are located some distance away from the active site. An analysis of the conformational dynamics of GCase suggests that mutations on these distal sites cause changes in the flexibility of active site residues despite their distance, indicating a dynamic communication network throughout the protein. The disruption of the long-distance dynamic coupling caused by missense mutations may provide a plausible general mechanistic explanation for biological dysfunction and disease.     

Isolation of indigenous Staphylococcus sciuri from chromium-contaminated paddy field and its application for reduction of Cr(VI) in rice plants cultivated in pots

Accumulation of Cr(VI) in rice seeds cultivated in Cr-contaminated soil of the Sundarbans (India) is an environmental problem. Cr(VI) concentration in this soil was 6.2 ± 0.3 mg/kg, whereas total chromium was 32.04 ± 1.60 mg/kg. A Cr(VI)-removing bacterium isolated from Cr-contaminated paddy field soil of Sundarbans was identified as Staphylococcus sciuri. Enrichment culture of S. sciuri was applied to pot cultivation of rice in Cr-contaminated soil. After 8 weeks, 71 ± 3% Cr(VI) (final concentration 2.15 ± 0.01 mg/kg) and 65 ± 2% total Cr removal (end concentration 11.3 ± 0.5 mg/kg) were attained in bacterium-treated soils. Growth parameters indicated healthy development of plants cultivated in bacterium-treated soils that was not observed in control plants. Total Cr removal attained in rice seeds of plants cultivated in bacterium-treated soils compared with control rice seeds was 78 ± 4%. Total Cr concentration in test seeds was 0.72 ± 0.05 mg/kg (World Health Organization [WHO] permissible limit: 1.30 mg/kg), whereas the same in control seeds was 3.27 ± 0.16 mg/kg. Cr(VI) reduction achieved in rice seeds cultivated in bacterium-treated soil compared with control rice seeds was 95 ± 5%. Cr(VI) concentration in rice seeds cultivated in treated soil was 0.050 ± 0.003 mg/kg, whereas the same in untreated control was 0.93 ± 0.05 mg/kg. Successful paddy field soil bioremediation by any Staphylococcus species was demonstrated for the first time.     

Structural and biochemical properties of lichenase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

The recombinant enzyme lichenase of size 30 kDa was over-expressed using E. coli cells and purified by immobilized metal ion affinity chromatography (IMAC) and size exclusion chromatography. The enzyme displayed high activity towards lichenan and β-glucan. The enzyme showed no activity towards carboxymethyl cellulose, laminarin, galactomannan or glucomannan. Surprisingly, affinity-gel electrophoresis on native-PAGE showed that the enzyme binds only glucomannan and not lichenan or β-glucan or other manno-configured substrates. The enzyme was thermally stable between the temperatures 60°C and 70°C. Presence of Cu²⁺ ions at a concentration of 5 mM enhanced enzyme activity by 10% but higher concentrations of Cu²⁺ (>25 mM) showed a sharp fall in the enzyme activity. Heavy metal ions Ni²⁺, Co²⁺ and Zn²⁺ did not affect the activity of the enzyme at low concentrations (0–10 mM) but at higher concentrations (>10 mM), caused a decrease in the enzyme activity. The crystals of lichenase were produced and the 3-dimensional structure of native form of enzyme was previously solved at 1.50 Å. Lichenase displayed (β/α)₈-fold a common fold among many glycoside hydrolase families. A cleft was identified that represented the probable location of active site.     

Two-dimensional gel-based alkaline proteome of the probiotic bacterium Lactobacillus acidophilus NCFM

Lactobacillus acidophilus NCFM (NCFM) is a well-documented probiotic bacterium isolated from human gut. Detailed 2D gel-based NCFM proteomics addressed the so-called alkaline range, i.e., pH 6-11. Proteins were identified in 150 of the 202 spots picked from the Coomassie Brilliant Blue stained 2D gel using MALDI-TOF-MS. The 102 unique gene products among the 150 protein identifications were assigned to different functional categories, and evaluated by considering a calculated distribution of abundance as well as grand average of hydrophobicity values. None of the very few available lactic acid bacteria proteome reference maps included the range of pI >7.0. The present report of such data on the proteome of NCFM fundamentally complements current knowledge on protein profiles limited to the acid and neutral pH range.     

Stabilizing Exposure of Conserved Epitopes by Structure Guided Insertion of Disulfide Bond in HIV-1 Envelope Glycoprotein

Entry of HIV-1 into target cells requires binding of the viral envelope glycoprotein (Env) to cellular receptors and subsequent conformational changes that culminates in fusion of viral and target cell membranes. Recent structural information has revealed that these conformational transitions are regulated by three conserved but potentially flexible layers stacked between the receptor-binding domain (gp120) and the fusion arm (gp41) of Env. We hypothesized that artificial insertion of a covalent bond will ‘snap’ Env into a conformation that is less mobile and stably expose conserved sites. Therefore, we analyzed the interface between these gp120 layers (layers 1, 2 and 3) and identified residues that may form disulfide bonds when substituted with cysteines. We subsequently probed the structures of the resultant mutant gp120 proteins by assaying their binding to a variety of ligands using Surface Plasmon Resonance (SPR) assay. We found that a single disulfide bond strategically inserted between the highly conserved layers 1 and 2 (C65-C115) is able to ‘lock’ gp120 in a CD4 receptor bound conformation (in the absence of CD4), as indicated by the lower dissociation constant (Kd) for the CD4-induced (CD4i) epitope binding 17b antibody. When disulfide-stabilized monomeric (gp120) and trimeric (gp140) Envs were used to immunize rabbits, they were found to elicit a higher proportion of antibodies directed against both CD4i and CD4 binding site epitopes than the wild-type proteins. These results demonstrate that structure-guided stabilization of inter-layer interactions within HIV-1 Env can be used to expose conserved epitopes and potentially overcome the sequence diversity of these molecules.     

The Role of Conformational Dynamics and Allostery in the Disease Development of Human Ferritin

Determining the three-dimensional structure of myoglobin, the first solved structure of a protein, fundamentally changed the way protein function was understood. Even more revolutionary was the information that came afterward: protein dynamics play a critical role in biological functions. Therefore, understanding conformational dynamics is crucial to obtaining a more complete picture of protein evolution. We recently analyzed the evolution of different protein families including green fluorescent proteins (GFPs), β-lactamase inhibitors, and nuclear receptors, and we observed that the alteration of conformational dynamics through allosteric regulation leads to functional changes. Moreover, proteome-wide conformational dynamics analysis of more than 100 human proteins showed that mutations occurring at rigid residue positions are more susceptible to disease than flexible residue positions. These studies suggest that disease-associated mutations may impair dynamic allosteric regulations, leading to loss of function. Thus, in this study, we analyzed the conformational dynamics of the wild-type light chain subunit of human ferritin protein along with the neutral and disease forms. We first performed replica exchange molecular dynamics simulations of wild-type and mutants to obtain equilibrated dynamics and then used perturbation response scanning (PRS), where we introduced a random Brownian kick to a position and computed the fluctuation response of the chain using linear response theory. Using this approach, we computed the dynamic flexibility index (DFI) for each position in the chain for the wild-type and the mutants. DFI quantifies the resilience of a position to a perturbation and provides a flexibility/rigidity measurement for a given position in the chain. The DFI analysis reveals that neutral variants and the wild-type exhibit similar flexibility profiles in which experimentally determined functionally critical sites act as hinges in controlling the overall motion. However, disease mutations alter the conformational dynamic profile, making hinges more loose (i.e., softening the hinges), thus impairing the allosterically regulated dynamics.