Expanding roles of cross-talk between hydrogen sulfide and nitric oxide under abiotic stress in plants.

Abiotic stress such as salt, heavy metals, drought, temperature, and others can affect plants from seed germination to seedling growth to reproductive maturity. Abiotic stress increases reactive oxygen species and lowers antioxidant enzymes in plants resulted the plant tolerance ability against stress conditions decrease. Hydrogen sulfide (H2S) and nitric oxide (NO) are important gasotransmitters involved in seed germination, photosynthesis, growth and development, metabolism, different physiological processes and functions in plants. In plants, various enzymes are responsible for the biosynthesis of both H2S and NO via both enzymatic and non-enzymatic pathways. They also mediate post-translation modification, such as persulfidation, and nitrosylation, which are protective mechanisms against oxidative damage. They also regulate some cellular signalling pathways in response to various abiotic stress. H2S and NO also stimulate biochemical reactions in plants, including cytosolic osmoprotectant accumulation, reactive oxygen species regulation, antioxidant system activation, K+ uptake, and Na+ cell extrusion or vacuolar compartmentation. In this review, we summarize how H2S and NO interact with each other, the function of both H2S and NO, the mechanism of biosynthesis, and post-translational modification under different abiotic stress. Our main emphasis was to find the cross-talk between NO and H2S and how they regulate genes in plants under abiotic stress.

Differential S-nitrosylation and characterization of purified S-nitrosoglutathione reductase (GSNOR) from Brassica juncea shows multiple forms of the enzyme.

S-nitrosoglutathione reductase (GSNOR). a master regulator of NO homeostasis, is a single-copy gene in most plants. In Lotus japonicus, two GSNOR isoforms were identified exhibiting similar kinetic properties but differential tissue-specific expressions. Previously, a genome-wide identification in Brassica juncea revealed four copies of GSNOR, each encoding proteins that vary in subunit molecular weights and pI. Here, we report multiple forms of GSNOR using 2D immunoblot which showed 4 immunopositive spots of 41.5 kDa (pl 5.79 and 6.78) and 43 kDa (pl 6.16 and 6.23). To confirm, purification of GSNOR using anion-exchange chromatography yielded 2 distinct pools (GSNOR-A & GSNOR-B) with GSNOR activities. Subsequently, affinity-based purification resulted in 1 polypeptide from GSNOR-A and 2 polypeptides from GSNOR-B. Size exclusion-HPLC confirmed 3 GSNORs with molecular weight of 87.48 ± 2.74 KDa (GSNOR-A); 87.36 ± 3.25 and 82.74 ± 2.75 kDa (GSNOR-B). Kinetic analysis showed Km of 118 ± 11 µM and Vmax of 287 ± 22 nkat/mg for GSNOR-A, whereas Km of 96.4 ± 8 µM and Vmax of 349 ± 15 nkat/mg for GSNOR-B. S-nitrosylation and inhibition by NO showed redox regulation of all BjGSNORs. Both purified GSNORs exhibited variable denitrosylation efficiency as depicted by Biotin Switch assay. To the best of our knowledge, this is the first report confirming multiple isoforms of GSNOR in B. juncea.

Identification of nitric oxide regulated low abundant myrosinases from seeds and seedlings of Brassica juncea.

Myrosinases constitute an important component of the glucosinolate-myrosinase system responsible for interaction of plants with microorganisms, insects, pest, and herbivores. It is a distinctive feature of Brassicales. Multiple isozymes of myrosinases are present in the vacuoles. Active myrosinases are also present in the apoplast and the nucleus however, the similarity or difference in the biochemical properties with the vacuolar myrosinases are not known. Here, we have attempted to isolate, characterize, and identify myrosinases from seeds, seedlings, apoplast, and nucleus to understand these forms. 2D-CN/SDS-PAGE coupled with western blotting and MS have shown low abundant myrosinases (65/70/72/75 kDa) in seeds and seedlings and apoplast & nucleus of seedlings to exist as dimers, oligomers, and as protein complex. Nuclear membrane associated form of myrosinase was also identified. The present study for the first time has shown enzymatically active myrosinase-alpha-mannosidase complex in seedlings. Both 65 and 70 kDa myrosinase in seedlings were S-nitrosated. Nitric oxide donor treatment (GSNO) led to 25% reduction in myrosinase activity which was reversed by DTT suggesting redox regulation of myrosinase. These S-nitrosated myrosinases might be a component of NO signalling in B. juncea.

Photosynthesis regulation, cell membrane stabilization and methylglyoxal detoxification seems major altered pathways under cold stress as revealed by integrated multi-omics meta-analysis.

Climate change has altered cold weather patterns, resulting in irregular cold weather conditions, and changing the global plant distribution pattern affecting plant development processes resulting in severe yield losses. Although molecular mechanisms and interconnections are quite well studied, a cumulative understanding of plant responses to cold stress (CS) is still lacking. Through meta-analysis, integration of data at the multi-omics level and its correlation with known physiological changes to map and understand the global changes in response to CS was made. Meta-analysis was conducted using the metafor R package program based on physiological parameters like relative electrolytic leakage, malondialdehyde, soluble sugar, proline and antioxidant enzymes activity. Proline and soluble sugars showed the highest (> 1.5 mean fold) change over control thus qualifying as global markers for studying CS. Surprisingly most up-regulated (> 15-fold) DEGs corresponded with the dehydrin family and glyoxalase superfamily proteins. Functional annotations of DEGs corresponded with photosynthesis and glycolysis pathway. Proteins responsible for cell signalling and increased soluble sugars were common in all the datasets studied thus correlating with the transcriptome and proteomic data. Proline and soluble sugars were positively regulated in all the metabolomics datasets. This study supported the earlier known players like proline and soluble sugars. Surprisingly, a new player glyoxalase seems to be contributing in CS. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01367-9.

Brassica juncea leaf cuticle contains xylose and mannose (xylomannan) which inhibit ice recrystallization on the leaf surface.

MAIN CONCLUSION: Conjugated sugars showed antifreeze activity in the cuticle by ice recrystallization inhibition rather than thermal hysteresis, enhancing freezing capacity at the surface of B. juncea leaves. Antifreeze biomolecules play a crucial role in mitigating the physical damage from frost by controlling extracellular ice crystal growth in plants. Antifreeze proteins (AFPs) are reported from the apoplast of different plants. Interestingly, there is no report about antifreeze properties of the cuticle. Here, we report the potential antifreeze activity in the Brassica juncea (BJ) leaf cuticle. Nano LC-MS/MS analysis of a cuticle protein enriched fraction (CPE) predicted over 30 putative AFPs using CryoProtect server and literature survey. Ice crystal morphology (ICM) and ice recrystallization inhibition (IRI) analysis of ABC supernatant showed heat and pronase-resistant, non-protein antifreeze activities as well as hexagonal ice crystals with TH of 0.17 °C and IRI 46%. The ZipTip processed ABC supernatant (without peptides) had no effect on TH activity, confirming a non-protein antifreeze molecule contributing to activity. To understand the origin and to confirm the source of antifreeze activity, cuticular membranes were isolated by pectinase and cellulase hydrolysis. FTIR analysis of the intact cuticle showed xylose, mannose, cellulose, and glucose. Xylanase and cellulase treatments of the ZipTip processed ABC supernatant led to an increase in sugar content and 50% loss in antifreeze activity. UV spectroscopy and NMR analysis supported the finding of FTIR and enzyme hydrolysis suggesting the contribution of xylose and mannose to antifreeze activity. By TLC analysis, conjugated sugars were found in the cuticle. This work has opened up a new research area where the antifreeze capacity needs to be established with regard to complete characterization and mechanism of action of the antifreeze carbohydrates (conjugated sugars) on the leaf surface.

N-Linked Glycoproteome Analysis of Diosorea alata Tuber Shows Atypical Glycosylation and Indicates Central Role of Glycosylated Proteins in Tuber Maturation.

Glycosylation is an important post translational modification in plants. First analysis of N-linked glycosylated proteins of Dioscorea alata using Concanavalin A lectin affinity chromatography enrichment coupled with label free quantification is presented. In total, 114 enriched glycoproteins were detected. Signal P and sub-cellular localization showed 42.2% of proteins to be secretory. These included peroxidases, endochitinases, calreticulin, calnexin, thaumatins and lipid transfer proteins. Gene Ontology and MapMan analysis predicted the enriched glycoproteins to be involved in processes essential for tuber maturation namely: signal transduction, lignification, protein trafficking, endoplasmic reticulum quality control and cell wall remodeling. This was supported by biochemical validation of the essential glycoproteins. Interestingly, out of the two dioscorin isoforms, Dio B was the only N-glycosylated form. In silico analysis showed O-glycosylation sites in the other form, Dio A suggesting its similarity with sporamin, the storage protein of sweet potato. Absence of signal peptide in Dio B and the presence of non-canonical motif hints towards its atypical glycosylation. The analysis revealed that N-glycosylation of Dio B isoform maintains the activities associated with Dioscorin at maturity and provides an overview of protein N-glycosylation, enriching the glycoproteome database of plants especially tubers.

Nitric oxide (NO) modulates low temperature-stress signaling via S-nitrosation, a NO PTM, inducing ethylene biosynthesis inhibition leading to enhanced post-harvest shelf-life of agricultural produce.

Low temperature (cold) stress is one of the major abiotic stress conditions affecting crop productivity worldwide. Nitric oxide (NO) is a dynamic signaling molecule that interacts with various stress regulators and provides abiotic stress tolerance. Stress enhanced NO contributes to S-nitrosothiol accumulation which causes oxidation of the -SH group in proteins leading to S-nitrosation, a post-translational modification. Cold stress induced in vivo S-nitrosation of > 240 proteins majorly belonging to stress/signaling/redox (myrosinase, SOD, GST, CS, DHAR), photosynthesis (RuBisCO, PRK), metabolism (FBA, GAPDH, TPI, SBPase), and cell wall modification (Beta-xylosidases, alpha-l-arabinogalactan) in different crop plants indicated role of NO in these important cellular and metabolic pathways. NO mediated regulation of a transcription factor CBF (C-repeat Binding Factor, a transcription factor) at transcriptional and post-translational level was shown in Solanum lycopersicum seedlings. NO donor priming enhances seed germination, breaks dormancy and provides tolerance to stress in crops. Its role in averting stress, promoting seed germination, and delaying senescence paved the way for use of NO and NO releasing compounds to prevent crop loss and increase the shelf-life of fruits and vegetables. An alternative to energy consuming and expensive cold storage led to development of a storage device called "shelf-life enhancer" that delays senescence and increases shelf-life at ambient temperature (25-27 °C) using NO donor. The present review summarizes NO research in plants and exploration of NO for its translational potential to improve agricultural yield and post-harvest crop loss. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01371-z.

Plant RABs: Role in Development and in Abiotic and Biotic Stress Responses.

Endosomal trafficking plays an integral role in various eukaryotic cellular activities and is vital for higher-order functions in multicellular organisms. RAB GTPases are important proteins that influence various aspects of membrane traffic, which consequently influence many cellular functions and responses. Compared to yeast and mammals, plants have evolved a unique set of plant-specific RABs that play a significant role in their development. RABs form the largest family of small guanosine triphosphate (GTP)-binding proteins, and are divided into eight sub-families named RAB1, RAB2, RAB5, RAB6, RAB7, RAB8, RAB11 and RAB18. Recent studies on different species suggest that RAB proteins play crucial roles in intracellular trafficking and cytokinesis, in autophagy, plant microbe interactions and in biotic and abiotic stress responses. This review recaptures and summarizes the roles of RABs in plant cell functions and in enhancing plant survival under stress conditions.

Brassica juncea leaf cuticle proteome analysis shows myrosinase protein, antifreeze activity, and post-translationally modified secretory proteins.

Plant cuticle, the site of perception of stress signals, is an extracellular hydrophobic barrier that covers the epidermis of the above-ground parts. This lipidic layer has been explored for its cutin and wax composition. However, reports on the cuticle proteins are scanty. Therefore, leaf cuticle proteins of Brassica juncea isolated using organic solvents (chloroform-methanol, 2:1(v/v)) were analyzed using gel based and quantitative shotgun proteomics. Out of 615 proteins identified, 27% (169) had signal peptides supporting extracellular localization. Bioinformatics tool, QuickGO predicted the involvement of these proteins in catabolism (21%), peptidase activity (13%), oxidoreductase (12%), defense response (9%), fatty acid binding (9%), nutrient reservoir activity (8%), chitin binding (7%) and lipid transport (2%). Myrosinase-catalyzed glucosinolate hydrolysis releases bioactive compounds, which contribute to plant defense. This system is termed as "mustard oil bomb". Myrosinase and its associating protein, GDSL esterase/lipase ESM1 (involved in cuticle structuring and defense) were detected in the cuticle. GDSL-esterase/lipase ESM1 and ß-glucanase (an antifreeze protein) showed in vitro activity. Analysis of cuticle extract by nanoliter osmometer-phase contrast microscopy detected antifreeze activity due to non-protein component. Post-translational modification analysis using PTM viewer predicted N-glycosylation (66%), N-terminal proteolysis (40%), and phosphorylation (32%) to be the dominant modification in the classical secretory proteins. N-glycosylation of myrosinase and GDSL esterase/lipase, ESM1 was confirmed by Con A affinoblotting. This study not only identified leaf cuticle proteins, but also laid the foundation for exploring the extracellular glucosinolate-myrosinase system, PTM crosstalk, and antifreeze activity as stress adaptive strategies in B. juncea.

Dioscorea Alata Tuber Proteome Analysis Uncovers Differentially Regulated Growth-associated Pathways of Tuber Development.

During its life cycle, the Dioscorea tuber undergoes multiple morphological and biochemical changes. To gain a better understanding of the metabolic changes associated with tuber growth, a stage-specific gel-free proteome analysis of four distinct morphological stages namely germinating tuber (S1), degrading tuber (S2), new tuber formation (S3) and tuber maturation (S4) was done and validated by principal component analysis. A comprehensive data set identifying 78.2% of the total 3,681 proteins was generated. PANTHER and KEGG MAPPER revealed both expected (carbohydrate metabolism and redox regulation) and novel biological processes (transcription factors and hormonal regulation) characteristic for each developmental stage. Higher abundance of the enzymes of ascorbate-glutathione cycle and carbohydrate metabolism was detected during tuber germination (S1) and tuber formation stages (S3) in comparison with the mature tuber. The presence of ethylene biosynthesis components during tuber formation hints toward its probable role in postharvest shelf life. The data set comprehensively describes the proteome of Dioscorea tuber and provides growth-specific markers for tuber germination (ascorbate peroxidase, monodehydroascorbate reductase, invertase) and tuber formation (sucrose synthase), which were validated by enzyme activity assays and Western blotting. The study provides information that may influence the direction of research for improving the productivity of this under-utilized and largely neglected crop.

Comparative fatty acid profiling of Indian seabuckthorn showed altitudinal gradient dependent species-specific variations.

The present study provides the first comparative fatty acid profiling of the three Indian seabuckthorn species, collected from varying altitudes (2900-4300 masl) of Trans-Himalayas (Hippophae rhamnoides, H. tibetana) and Sikkim Himalayas (H. salicifolia) regions. Gas chromatography-mass spectrometry analysis showed variability in fatty acid composition of different seabuckthorn populations. Sikkim populations showed higher (1.28-1.6 folds) palmitic acid than Trans-Himalayan populations which possess higher linoleic (1.3-1.5 folds) and linolenic (1.6-1.8 folds) acids. Interestingly, a strong altitudinal gradient associated positive correlation was observed with the degree of unsaturation and PUFA content while negative correlation was observed with saturated fatty acids content of different seabuckthorn populations. H. salicifolia collected from Sikkim showed healthy ω-6:ω-3 ratio (closer to 1:1) of functional lipids exhibiting its better nutraceutical potential than other commonly used seed oils. Interestingly, H. tibetana from Losar showed higher (5.81) degree of unsaturation than Sikkim populations (3.5) suggesting its better stress tolerance trait. Chemo-taxonomic diversity analysis also formed two broad clusters of Trans-Himalayan and Sikkim populations which correlated with earlier taxonomic studies.

Ecophysiolomic analysis of stress tolerant Himalayan shrub Hipppophae rhamnoides shows multifactorial acclimation strategies induced by diverse environmental conditions.

Climatic fluctuations are a major global concern, affecting the agronomic productivity of plants. Hippophae rhamnoides a naturally growing stress tolerant Himalayan shrub was chosen to understand its stress hardiness mechanism. Comparative proteomic and biochemical analysis were done for pooled berry populations (HrB13 and HrB14) growing in two different environmental conditions. HrB13, growing under sub-optimal environmental conditions exhibited differential abundance of stress responsive proteins, which were the rate limiting enzymes associated with stress-responsive metabolic pathways, including Xanthine dehydrogenase (reactive oxygen species [ROS] signaling), Farnesyl diphosphate synthase (phenylpropanoid pathway), endosomal BRO-1 domain protein (ultraviolet [UV]-light stress), Phosphofructokinase (sugar metabolism) and Ubiquitin thioesterase (protein alterations). Biochemical investigations showed a positive correlation between proteomic plasticity (HrB13) and 1.6 to 15-fold accumulation of downstream adaptive metabolic signatures like enzymes and antioxidants involved in ROS scavenging pathways (Catalase, Ascorbate peroxidase, Glutathione reductase, ascorbate and glutathione content), secondary metabolites (phenolics, flavonoids, carotenoids) and polyunsaturated fatty acids (∝ - linolenic acid and linoleic acid). Interactome and KEGG pathway analysis also supported interactions of differentially accumulated proteins with stress-responsive signaling components involved in physiological pathways associated with stress tolerance. This is the first 'ecophysiolomics' study, showing the response of seabuckthorn to multiple stress conditions via activation of multifactorial acclimation strategies leading to morphological, metabolic and physiological modifications, resulting in dark orange berries in HrB13. Higher accumulation of omega-6 fatty acids, carotenoids and ascorbate during suboptimal growth conditions, provides exciting prospects for enhancing pharmaceutical properties of seabuckthorn berries, emphasizing need to analyze diversity of plant signaling mechanisms under changing climate conditions.

Analysis of temporally evolved nanoparticle-protein corona highlighted the potential ability of gold nanoparticles to stably interact with proteins and influence the major biochemical pathways in Brassica juncea.

Nanoparticles (NPs) are known to adsorb proteins from their surroundings, forming NP-protein corona, which determines their fate, distribution, and effects, yet no information of protein corona (PC) has been gathered in the plants so far. Here we report, the analysis of temporally evolved (2 h-36 h) AuNP-protein coronas formed with Brassica juncea leaf crude protein & nuclear-enriched fraction. Protein coronas were characterized by the techniques including SDS PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), spectrophotometry, dynamic light scattering, zeta potential measurements, and Nano LC-MS/MS. Data analysis revealed the formation of two regimes (Regime I from 2 to 8 h & regime II from 16 to 36 h). Interestingly, coated AuNPs had approx. 30% higher zeta potential than pristine AuNPs after 36 h of interactions. The increase in hydrodynamic radii and adsorbed protein concentrations were consistent with the evolution of zeta potential, indicating the probable role of proteins in providing the better stability of AuNPs. MS analysis identified 97 proteins from regime I (soft corona) and 181 proteins from regime II (hard corona) of crude PC. On the other hand, 282 and 308 proteins were identified from nuclear soft and hard corona respectively, indicating better affinity of nuclear proteins. Besides, the high-affinity proteins (fold change ≥5) were found to be rich in lysine residues showing their involvement in promoting the adsorption. Notably, 27% of regime II corona proteins of the crude protein fraction were from energy-yielding pathways highlighting the potential ability AuNPs to influence the yield in Brassica juncea.

Two ICE isoforms showing differential transcriptional regulation by cold and hormones participate in Brassica juncea cold stress signaling.

C-repeat binding factor (CBF) dependent cold stress signaling cascade is well studied in the model plant arabidopsis but is relatively lesser studied in the crop plants. In the present study, two novel isoforms of an upstream regulator of CBF, Inducer of CBF expression (ICE), BjICE46 (1314 bp, accession number HQ446510) and BjICE53 (1494 bp, accession number HQ857208) were isolated from Brassica juncea seedlings. Genomic clones of both the isoforms (accession numbers HQ433510 and JX571043) showed three introns, out of which one intron was spanning the bHLH (basic helix-loop-helix) domain. Interestingly, the constitutive expression of BjICE53 was 21 fold higher than BjICE46. Real time quantitative expression (RT-qPCR) showed BjICE53 to be cold induced but non-responsive to phytohormones. Interestingly, BjICE46 was salinity stress induced and showed upregulation with methyl jasmonate (MeJa) and abscisic acid (ABA). This was supported by the presence of ABA, MeJa and defense related cis- acting regulatory elements in the promoter region of BjICE46. The downstream transcription factor BjCBF (645 bp) was also isolated. The promoter region of BjCBF showed three E-boxes, the binding site for ICE. BjCBF was expressed and purified from E. coli and binding of purified BjCBF with the DRE/CRT elements (present in the promoter of cold responsive genes) was EMSA confirmed. Overall, this study shows that ICE-CBF pathway is conserved in Brassica juncea along with the differential regulation of the ICE isoforms indicating cross-talk between cold and defense signaling.

Single-step purification and characterization of antifreeze proteins from leaf and berry of a freeze-tolerant shrub seabuckthorn (Hippophae rhamnoides).

Seabuckthorn is a freeze-tolerant Himalayan shrub, capable of withstanding temperatures below -40°C. Antifreeze proteins prevent freezing associated damage by restricting ice crystals growth. In the present study, homogenous purification of two antifreeze proteins (41 and 39 kDa) from Hippophae rhamnoides leaf and one (41 kDa) from berry was performed using ice-affinity chromatography. MS identification and Basic Local Alignment Search Tool search showed homology of berry antifreeze proteins with disease resistance protein while leaf antifreeze proteins showed similarity with transmembrane protein (39 kDa) and low temperature induced protein (41 kDa) suggesting their role in cold stress signalling. Hexagon shaped ice crystals (Nanoliter osmometer) and ice recrystallization inhibition assay (Splat assay) confirmed higher ice recrystallization inhibition activity of purified leaf (2.5 fold decrease in mean ice crystal size) and berry (2.1 fold decrease) antifreeze proteins. String interactome analysis showed interaction of antifreeze proteins with cold stress modulated targets including pathogenesis related proteins. This probably is the first report of antifreeze proteins purification from naturally growing seabuckthorn. Further validation of these targets may open gates for commercial utilization of this plant growing abundantly in Himalayan regions of India, for crop improvement of freeze susceptible crops or biomedical applications like cryopreservation of tissues and cells.

Single pot synthesized gold nanoparticles using Hippophae rhamnoides leaf and berry extract showed shape-dependent differential nanobiotechnological applications.

A facile one-pot green synthesis of gold nanoparticles (AuNPs) with different geometries was achieved using an underutilized Himalayan bioresource Hippophae rhamnoides. Aqueous leaf (LE) and berry extracts (BE) showed rapid synthesis of monodispersed spherical LEAuNPs (27 ± 3.2 nm) and anisotropic BEAuNPs (55 ± 4.5 nm) within 2 and 15 min, respectively. The Fourier-transform infrared (FTIR) spectroscopy showed involvement of polyphenolics/flavonoids in AuNPs reduction. LE AuNPs (IC50 49 µg) exhibited higher antioxidant potential than BE AuNPs (IC50 57 µg). Both BE nanotriangles and LE nanospheres exhibited cytotoxicity against Jurkat cell lines. These nanocatalysts also exhibited effective (80-99%) reductive degradation of structurally different carcinogenic azo dyes. Kinetic studies revealed that BE nanotriangles exhibited higher catalytic efficiency (14-67%) than LE nanospheres suggesting shape-dependent regulation of biological activities. The gas chromatography-mass spectrometry (GC-MS) analysis confirmed conversion of toxic methyl orange dye to non-toxic intermediates. Probable degradation mechanism involving adsorption and catalytic reduction of azo bonds was proposed. The present synthesis protocol provided a facile and energy saving procedure for rapid synthesis of highly stable nanoparticles with significant antioxidant and anticancer potential. This is the first report of H. rhamnoides-mediated green synthesis of multipurpose AuNPs as antioxidant, anticancer and nanocatalytic agents for treatment of dye contaminated waste water and future therapeutic applications.

Green silver nanoparticles from novel Brassicaceae cultivars with enhanced antimicrobial potential than earlier reported Brassicaceae members.

In the present study, we report perhaps for the first time the use of novel varieties of Brassica oleracea var. botrytis and Raphanus sativus as potential bioreductant, to synthesize highly stable silver nanoparticles (AgNPs, no aggregation observed for six months), which is a significant finding as plant extract-directed AgNPs are intrinsically unstable and tend to aggregate. The reduction of Ag+ to Ag0 nanostructures was confirmed using UVVis spectroscopy showing SPR spectra at 400-435 nm. Nanosight and transmission electron microscope (TEM) analysis showed monodisperse spherical AgNPs (4-18 nm). Fourier transform infrared spectroscopy (FTIR) analysis revealed that the polyphenolics and other secondary metabolites including glucosinolates in the aqueous extracts may act as reducing/capping agent for the nanoparticle synthesis. X-ray diffraction (XRD) confirmed the face centered cubic crystalline (fcc) structure of AgNPs. Controlled synthesis of AgNPs was achieved by varying experimental parameters (AgNO3 concentration, extract volume, pH and temperature). These AgNPs exhibited strong antibacterial activity at significantly lower concentration (5 ppm) against both Gram negative (Escherichia coli, Myroides, Psuedomonas aeruginosa) and Gram positive (Kocuria and Promicromonospora) bacteria. In the present study, the green AgNPs showed (10-30%) better antimicrobial efficacy than chemical AgNPs and AgNPs from other Brassicaceae members. These green AgNPs may have promising application in nano-drug formulation to combat bacterial infections, in future.

A novel class I Chitinase from Hippophae rhamnoides: Indications for participating in ICE-CBF cold stress signaling pathway.

Plant chitinases are the members of PR (Pathogenesis related) proteins family and protect plants from biotic and abiotic stress. A novel chitinase HrCHI1 (Accession number JQ289153) of 954bp ORF encoding 317 amino acids protein was cloned, expressed and characterized from seabuckthorn, a cold/freeze tolerant shrub. The 3D structure (predicted with I-TASSER server) showed highest homology with Oryza sativa class I chitinase (PDB 2dkvA). Putative promoter region (obtained by genome walking) showed GCC box, E-boxes, the binding site for bHLH proteins and DRE elements, the CBF (C-repeat binding factor) binding site besides TATA and CAAT boxes. The gel shift assay with the nuclear extract indicated that the HrCHI1 might be participating in CBF/ERF dependent cold stress signaling pathway. The quantitative transcript profiling supported this observation as cold induced expression of HrCBF peaked earlier (at 1h) while HrCHI1 peaked latter (after 3h) indicating HrCHI1 expression might be induced by HrCBF. Further, HrCHI1 expression was methyl jasmonate (MeJa) dependent and salicylic acid (SA) independent. HrCHI1 was expressed in E. coli and purified using chitin affinity chromatography. It showed 512U/mg chitinase hydrolytic activity and resolved as a 34kDa spot with a slightly basic pI (8.5) on a 2-D gel. The E. coli cells containing recombinant chitinase showed higher rate of growth in cold in comparison with the cells containing the empty vector. In conclusion, we have isolated and characterized a cold responsive basic class I chitinase which is regulated by MeJa and seems to be functioning via CBF/ERF dependent cold stress signaling pathway.