Juncea Seedlings Research Articles

Role of Se(VI) in counteracting oxidative damage in Brassica juncea L. under Cr(VI) stress

Chromium (Cr) is considered to be one of the major environmental hazards and poses a threat to both plant and animal health. Selenium (Se), however, has been recognized as an essential micronutrient in plants. To understand the role of Se(VI) in oxidative stress management and regulation of antioxidative defence mechanism against heavy metal stress, the seedlings of Brassica juncea L. were raised in Petri plates containing nutrient media supplemented with only with Se(VI) and Cr(VI), or their combination. It was observed that of Cr(VI) causes an increase in reactive oxygen species (ROS) in the seedlings leading to oxidative stress. Histological studies using confocal and visible microscopy confirmed the biochemical results. Supplementation of up to 4 µM of Se(VI) to media containing 300 µM of Cr(VI) reduced the contents of ROS and increased enzymatic and non-enzymatic antioxidants in the seedlings. At a concentration of 6 µM, however, Se(VI) was toxic. The results suggested that at appropriate concentrations, the exogenous application of Se(VI) enabled the B. juncea seedlings to counteract the effects of Cr(VI), thereby increasing the resistance of plants.

Pre-sowing Seed Treatment with 24-Epibrassinolide Ameliorates Pesticide Stress in Brassica juncea L. through the Modulation of Stress Markers.

The present experiment was designed to assess the effects of seed soaking with 24-epibrassinolide (EBR) on the physiology of Brassica juncea L. seedlings grown under imidacloprid (IMI) toxicity. Application of EBR increased the length of seedlings, dry weight, and pigment contents, polyphenols, total phenols, and organic acids under IMI toxicity. The expression of genes coding key enzymes of pigment, phenols, polyphenols, and organic acid biosynthetic pathways was also studied including CHLASE (chlorophyllase), PSY (phytoene synthase), CHS (chalcone synthase) and PAL (phenylalanine ammonialyase), CS (citrate synthase), SUCLG1 (succinyl Co-A ligase,), SDH (succinate dehydrogenase), FH (fumarate hydratase), MS (malate synthase). Multiple linear regression (MLR) analysis revealed that IMI application regressed negatively on seedling length, dry weight and total chlorophyll content. However, EBR seed treatment regressed positively on all the parameters studied. Moreover, interaction between IMI and EBR showed positive regression for growth parameters, content of pigments, total polyphenol, total phenol and malate, and expression of PSY and PAL. Negative interactions were noticed for the contents of fumarate, succinate and citrate, and expression of CHS and all genes studied related to organic acid metabolism. In conclusion, EBR enhanced the growth and contents of all studied metabolites by regulating the gene expression of B. juncea seedlings under IMI stress.

Co-application of 6-ketone type brassinosteroid and metal chelator alleviates cadmium toxicity in B. juncea L.

Plant growth regulator-assisted phytoremediation has been assessed as a novel strategy to improve phytoremediation potential of plants. In the present work, potential of castasterone, a plant growth regulator, combined with citric acid was explored for phytoremediation of cadmium in Brassica juncea seedlings. The seedlings were raised under controlled laboratory conditions for 7days. Results revealed that 0.6mM cadmium exposure induced toxicity in the seedlings, which was reflected through root growth inhibition, accumulation of hydrogen peroxide and malondialdehyde, and loss of cell viability. Pre-sowing treatment of castasterone supplemented with citric acid enhanced cadmium accumulation in the roots (from 752μg/g DW to 1192μg/g DW) and shoots (from 88μg/g DW to 311μg/g DW) and also improved root length, shoot length, fresh weight, and dry weight of seedlings by 81, 17, 39, and 35%, respectively. The co-application reduced malondialdehyde accumulation by 39% and reduced oxidative stress by enhancing the activities of antioxidant enzymes (superoxide dismutase, guaiacol peroxidase, catalase, ascorbate peroxidase, dehydroascorbate, glutathione reductase, glutathione peroxidase, glutathione-S-transferase, polyphenol oxidase), maximum enhancement (82%) being in polyphenol oxidase. Similarly, the contents of water- and lipid-soluble antioxidants were found to increase by 31 and 4%, respectively. Confocal microscopy revealed enhanced content of NO. Results suggested that binary combination of castasterone and citric acid is helpful in improving cadmium accumulation and ameliorating metal toxicity in B. juncea seedlings.

Carbon: Nitrogen Interaction Regulates Expression of Genes Involved in N-Uptake and Assimilation in Brassica juncea L.

In plants, several cellular and metabolic pathways interact with each other to regulate processes that are vital for their growth and development. Carbon (C) and Nitrogen (N) are two main nutrients for plants and coordination of C and N pathways is an important factor for maintaining plant growth and development. In the present work, influence of nitrogen and sucrose (C source) on growth parameters and expression of genes involved in nitrogen transport and assimilatory pathways was studied in B. juncea seedlings. For this, B. juncea seedlings were treated with four combinations of C and N source viz., N source alone (-Suc+N), C source alone (+Suc-N), with N and C source (+Suc+N) or without N and C source (-Suc-N). Cotyledon size and shoot length were found to be increased in seedlings, when nitrogen alone was present in the medium. Distinct expression pattern of genes in both, root and shoot tissues was observed in response to exogenously supplied N and C. The presence or depletion of nitrogen alone in the medium leads to severe up- or down-regulation of key genes involved in N-uptake and transport (BjNRT1.1, BjNRT1.8) in root tissue and genes involved in nitrate reduction (BjNR1 and BjNR2) in shoot tissue. Moreover, expression of several genes, like BjAMT1.2, BjAMT2 and BjPK in root and two genes BjAMT2 and BjGS1.1 in shoot were found to be regulated only when C source was present in the medium. Majority of genes were found to respond in root and shoot tissues, when both C and N source were present in the medium, thus reflecting their importance as a signal in regulating expression of genes involved in N-uptake and assimilation. The present work provides insight into the regulation of genes of N-uptake and assimilatory pathway in B. juncea by interaction of both carbon and nitrogen.

Mitigation of NaCl Stress by Arbuscular Mycorrhizal Fungi through the Modulation of Osmolytes, Antioxidants and Secondary Metabolites in Mustard (Brassica juncea L.) Plants.

Present work was carried out to investigate the possible role of arbuscular mycorrhizal fungi (AMF) in mitigating salinity-induced alterations in Brassica juncea L. Exposure to NaCl stress altered the morphological, physio-biochemical attributes, antioxidant activity, secondary metabolites and phytohormones in the mustard seedlings. The growth and biomass yield, leaf water content, and total chlorophyll content were decreased with NaCl stress. However, AMF-inoculated plants exhibited enhanced shoot and root length, elevated relative water content, enhanced chlorophyll content, and ultimately biomass yield. Lipid peroxidation and proline content were increased by 54.53 and 63.47%, respectively with 200 mM NaCl concentration. Further increase in proline content and decrease in lipid peroxidation was observed in NaCl-treated plants inoculated with AMF. The antioxidants, superoxide dismutase, ascorbate peroxidase, glutathione reductase, and reduced glutathione were increased by 48.35, 54.86, 43.85, and 44.44%, respectively, with 200 mM NaCl concentration. Further increase in these antioxidants has been observed in AMF-colonized plants indicating the alleviating role of AMF to salinity stress through antioxidant modulation. The total phenol, flavonoids, and phytohormones increase with NaCl treatment. However, NaCl-treated plants colonized with AMF showed further increase in the above parameters except ABA, which was reduced with NaCl+AMF treatment over the plants treated with NaCl alone. Our results demonstrated that NaCl caused negative effect on B. juncea seedlings; however, colonization with AMF enhances the NaCl tolerance by reforming the physio-biochemical attributes, activities of antioxidant enzymes, and production of secondary metabolites and phytohormones.

The chemical toxicity of cesium in Indian mustard (Brassica juncea L.) seedlings

To distinguish between the radiological and chemical effects of radiocesium, we study the chemical toxicity of cesium in the seedlings of Indian mustard (Brassica juncea L.). In this study, the experiment was designed in two factors and five levels random block design to investigate the interaction effects of Cs and K. Results showed that excessive Cs was one of the main factors influence the growth of Brassica juncea seedlings. And the toxicity of Cs in Brassica juncea is likely to be caused by Cs interacts with K-binding sites in essential K-dependent protein, either competes with K for essential biochemical functions, causing intracellular metabolic disturbance. To test the hypothesis that the toxicity of Cs might cause intracellular metabolic disturbance, next-generation sequencing (NGS)-based Illumina paired-end Solexa sequencing platform was employed to analysis the changes in gene expression, and understand the key genes in B. juncea seedlings responding to the toxicity of Cs. Based on the assembled de novo transcriptome, 2032 DEGs that play significant roles in the response to the toxicity of Cs were identified. Further analysis showed that excessive Cs is disturbance the auxin signal transduction pathway, and inhibited the indoleacetic acid-induced protein (AUX/IAA) genes expression eventually lead the seedlings growth and development be inhibited. The results suggest that disturbances to tryptophan metabolism might be linked to changes in growth.

Alleviation of selenium toxicity in Brassica juncea L.: salicylic acid-mediated modulation in toxicity indicators, stress modulators, and sulfur-related gene transcripts.

The present work reveals the response of different doses of selenium (Se) and alleviating effect of salicylic acid (SA) on Se-stressed Brassica juncea seedlings. Selenium, a micronutrient, is essential for both humans and animals but is toxic at higher doses. Its beneficial role for the survival of plants, however, is still debatable. On the other hand, SA, a phenolic compound, is known to have specific responses under environmental stresses. Experiments were conducted using leaves of hydroponically grown seedlings of Pusa bold (PB) variety of B. juncea, treated with different concentrations of Se (50, 150, 300μM) for 24- and 96-h exposure times. Increasing Se concentrations inhibited growth and, caused lipid peroxidation, concomitantly increased stress modulators (proline, cysteine, SOD, CAT) along with sulfur-related gene transcripts (LAST, APS, APR, GR, OASL, MT-2, PCS) in Brassica seedlings. On the basis of the above studied parameters, maximum inhibition in growth was observed at 300μM Se after 96-h exposure time. Further, co-application of SA along with 300μM Se helped to mitigate Se stress, as shown by improved levels of growth parameters, toxicity indicators (chlorophyll, protein, MDA), stress modulators (proline, cysteine, SOD, and CAT), and expression of sulfur-related genes as compared to Se-treated seedlings alone. Altogether, this study revealed that Se + SA combinations improved seedling morphology and were effective in alleviation of Se stress in PB variety of B. juncea.

Can the use of natural biostimulants be a potential means of phytoremediating contaminated soils from goldmines in South Africa?

ABSTRACTBiostimulants offer great potential in improving phytoremediation of contaminated soils. In the current greenhouse-based study, Brassica juncea seedlings grown on soils collected from Krugersdorp Goldmine and the adjourning areas (a Game Reserve and private farmland) were supplemented with different biostimulants (Kelpak® = KEL, vermicompost leachate = VCL, smoke-water = SW). Indole-3-butyric acid (IBA) was included in the study for comparative purposes because these biostimulants are known to enhance rooting. Prior to the pot trial, concentrations of elements in the three soil types were determined using Inductively Coupled Plasma-Optical Emission Spectroscopy. Plants were harvested after 105 days and the growth and concentrations of elements in the various plant organs were determined. TheB. juncea seedlings with and without biostimulants did not survive when growing in soil from the Krugersdorp Goldmine. The Game Reserve and private farmland soils supplemented with KEL produced the highest plant biomass and the lowest accumulation of metals in the organs of B. juncea. High concentrations (>13 000 mg kg−1) of zinc and aluminium were quantified in the roots of IBA-supplemented soils from the Game Reserve. Generally, IBA and SW enhanced the phytoremediation of B. juncea due to elevated levels of elements that accumulated in their different organs.

Biotic elicitors and mechanical damage modulate glucosinolate accumulation by co-ordinated interplay of glucosinolate biosynthesis regulators in polyploid Brassica juncea

Glucosinolates are nitrogen and sulfur containing secondary metabolites found mainly in the Brassicaceae. They function as plant defense compounds against a broad spectrum of pathogens and pests. Since these molecules form part of the plant defense mechanism, glucosinolate biosynthesis may be modulated by environmental signals leading to activation of a biological stress response. In the current study, we have mimicked such conditions by exogenously applying biotic elicitors such as methyl jasmonate, salicylic acid, glucose and mechanical injury in Brassica juncea seedling over a time course experiment. We found that total glucosinolates over-accumulated under these stress conditions with maximum accumulation observed 24h post treatment. Indole glucosinolates like 1-methoxy-indol-3-ylmethyl and its precursor indol-3-methyl glucosinolates showed a more significant induction compared to aliphatic glucosinolates thereby suggesting a prominent role of indole glucosinolates during plant defense response in B. juncea seedlings. In contrast, the higher amounts of aliphatic glucosinolates were less regulated by the tested biotic elicitors in B. juncea. Expression profiling of multiple homologs of key transcriptional regulators of glucosinolate biosynthesis further showed that a complex interplay of these regulators exists in polyploid B. juncea where they exert co-ordinated and overlapping effects toward altering glucosinolate accumulation. This study has a significant role toward understanding and augmenting plant defense mechanisms in B. juncea, a globally important oilseed crop of genus Brassica.

Role of Heme Oxygenase1 in Alteration of Antioxidant Defense Responses of Brassica Juncea (L.) Czern. under Salt Stress Condition

Economically important crops are frequently facing many stresses, such as salt stress. They have developed an antioxidant defense network for preventing and repairing oxidative damage caused by salt stress. In this study, Brassica juncea seedlings were subjected to salt stress (NaCl) for time course analysis (1 st -5 th day) of heme oxygenase1 (HO1) activity along with other antioxidant enzymes. Treatment of salt induced lipid peroxidation and H2O2 content in root tissue as days of treatment increased. However, 1 st and 2 nd day of treatment did not modify these parameters, were similar to control values. HO1, Ascorbate peroxidase (APX), Peroxidase (POD) and Glutathione reductase (GR) activities were highest at 5 th day of treatment. Gene expression pattern of HO1 was similar as shown in biochemical analysis. These results showed that HO1 along with other antioxidant enzymes play an essential protective role against salt stress in B. juncea seedlings.

Antioxidant activity of heme oxygenase 1 in Brassica juncea (L.) Czern.(Indian mustard) under salt stress

Antioxidant enzymes play a protective role in plants under oxidative stress. The present study explores the role of antioxidant system heme oxygenase1 (HO1) in Brassica juncea (L.) Czern. in salt stress conditions. B. juncea seedlings were treated with different NaCl concentrations (0-200 mM) for 5 days. Salt stress elicited the highest response at 150 mM NaCl concentration; at this level, gene expression of HO1 was also upregulated. Apart from HO1, ascorbate peroxidase (APX), peroxidase (POD), and glutathione reductase (GR) also showed their highest activity at 150 mM NaCl concentration. The study shows that HO1 is indispensable in B. juncea for overcoming the oxidative stress caused by salinity. Understanding the intricacy of the defense network, including HO1, against salt stress will be useful for future research into developing salt-tolerant varieties.

The significance of glucosinolates for sulfur storage in Brassicaceae seedlings.

Brassica juncea seedlings contained a twofold higher glucosinolate content than B. rapa and these secondary sulfur compounds accounted for up to 30% of the organic sulfur fraction. The glucosinolate content was not affected by H2S and SO2 exposure, demonstrating that these sulfur compounds did not form a sink for excessive atmospheric supplied sulfur. Upon sulfate deprivation, the foliarly absorbed H2S and SO2 replaced sulfate as the sulfur source for growth of B. juncea and B. rapa seedlings. The glucosinolate content was decreased in sulfate-deprived plants, though its proportion of organic sulfur fraction was higher than that of sulfate-sufficient plants, both in absence and presence of H2S and SO2. The significance of myrosinase in the in situ turnover in these secondary sulfur compounds needs to be questioned, since there was no direct co-regulation between the content of glucosinolates and the transcript level and activity of myrosinase. Evidently, glucosinolates cannot be considered as sulfur storage compounds upon exposure to excessive atmospheric sulfur and are unlikely to be involved in the re-distribution of sulfur in B. juncea and B. rapa seedlings upon sulfate deprivation.

Study on the correlation between copper oxide nanoparticles induced growth suppression and enhanced lignification in Indian mustard (Brassica juncea L.)

In this study, the morphological, physiological and molecular level effects of copper oxide nanoparticles (CuONPs) were studied in an economically important oil seed crop Brassica juncea L. The possible involvement of lignification on shoot–root growth retardation was also studied. The seedlings were exposed to 0, 20, 50, 100, 200, 400 and 500mg/L of CuONPs in semi-solid half strength Murashige and Skoog medium under controlled growth chamber conditions for 14 days. Exposure to CuONPs resulted in suppression of shoot-growth, reduction in total chlorophyll and carotenoids contents as well modification of root system architecture such as shortening of primary and lateral roots. Significant increases in hydrogen peroxide formation, peroxidase enzyme activity and lignification of shoots and roots were observed. The lipid peroxidation levels increased significantly in shoots and roots of B. juncea seedlings. Phloroglucinol-HCl staining revealed enhanced lignification of shoot and roots. Gene expression studies revealed significant activation of CuZn superoxide dismutase (CuZnSOD) in roots at all concentrations of CuONPs exposure. In shoots significant up-regulation of CuZnSOD gene was observed upon exposure to 100, 200 and 400mg/L of CuONPs exposure. However no change in the expression levels of MnSOD gene was observed in both stem and roots. The expression of catalase (CAT) and ascorbate peroxidase (APX) genes were also not changed in shoots. However, significant inhibition of CAT and APX genes were observed in roots of B. juncea plants under exposure to 100, 200, 400 and 500mg/L of CuONPs exposure. The SOD enzyme activity significantly increased in roots under exposure to 50–500mg/L of CuONPs and in shoots as a result of exposure to 100–500mg/L of CuONPs. The APX activity significantly decreased in roots upon exposure to 50–500mg/L of CuONPs. In shoots, the APX activity significantly decreased upon exposure to 200–500mg/L of CuONPs.

A Genome-Wide Perspective of miRNAome in Response to High Temperature, Salinity and Drought Stresses in Brassica juncea (Czern) L

Micro RNAs (miRNAs) are involved in diverse biological processes including adaptive response towards abiotic stresses. To unravel small RNAs and more specifically miRNAs that can potentially regulate determinants of abiotic stress tolerance, next generation sequencing of B. juncea seedlings subjected to high temperature, high salt and drought conditions was carried out. With the help of UEA sRNA workbench software package, 51 conserved miRNAs belonging to 30 miRNA families were identified. As there was limited genomic information available for B. juncea, we generated and assembled its genome sequence at a low coverage. Using the generated sequence and other publically available Brassica genomic/transcriptomic resources as mapping reference, 126 novel (not reported in any plant species) were discovered for the first time in B. juncea. Further analysis also revealed existence of 32 and 37 star sequences for conserved and novel miRNAs, respectively. The expression of selected conserved and novel miRNAs under conditions of different abiotic stresses was revalidated through universal TaqMan based real time PCR. Putative targets of identified conserved and novel miRNAs were predicted in B. rapa to gain insights into functional roles manifested by B. juncea miRNAs. Furthermore, SPL2-like, ARF17-like and a NAC domain containing protein were experimentally validated as targets of miR156, miR160 and miR164 respectively. Investigation of gene ontologies linked with targets of known and novel miRNAs forecasted their involvement in various biological functions.

28-Homobrassinolide-Induced Exaggerated Growth, Biochemical Molecular Aspects of Brassica Juncea L. RLM-619 Seedlings under High Temperature Stress

The present study was pertaining to study the mechanism and signaling of BRs under high temperature stress in Brassica juncea L. plants. Results advised that high temperature stress reduced the shoot length, root length, fresh weight of the plants but 28-homobrassinosteroids (28-homoBL) treatment reduced the toxic effect of high temperature stress by improving the same of B. juncea L. plants. To ascertain these free radical scavenging enzymes (superoxide dismutase (SOD), Catalase (CAT), Peroxidase (POD), Ascorbate peroxidase (APOX), Polyphenol oxidase (PPO) and Indole acetic acid oxidase (IAAO) and lipid oxidation (MDA) were assayed along with photosynthetic pigments, total carbohydrates, reducing sugars and non-reducing sugars. All studied scavenging enzymes activities were ameliorated by the homoBL treatments. The pigments decreased under temperature stress were also protected by homoBL treatments. Proteins profiling by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) were also studied to know the behavior of proteins under temperature stress as well as with the treatment of homoBL. Protein bands disappeared in temperature stress while in the presence of homoBL treatment existing proteins displayed more accumulation leading to the appearance of new bands. Native-PAGE of SOD, CAT and POD suggested the presence of in isoenzymic forms in cells. Application of different concentration of homoBL increased intensities of these isoenzymes under temperature and normal condition as compared to control untreated seedlings. The gene expression of SOD and CAT also suggested that synthesis these enzymes were decreased under high temperature stress but ameliorated with the treatment of homoBL. Present study demonstrated that 28-homoBL have stress-ameliorative properties in B. juncea seedlings exposed to high temperature stress by improving seedling growth and enhancing protein content as well as activities of SOD, CAT, APOX, PPO and IAA Oxidase. Further, it was suggested by decreasing the MDA content after the treatments of 28-homoBL. This study demonstrate the culmination of BR’s as an anti-stressor for protection of plant exposed to high temperature stress but extensive studies are still needed to know the various aspects related to stress and role of brassinosteroids in regulating them at molecular and signaling level.

Histochemical Detection of Superoxide and H2O2 Accumulation in Brassica juncea Seedlings

Plant cells continually produce reactive oxygen species (ROS) as a by-product of aerobic metabolism. Increased production of ROS occurs under unfavorable conditions imposed by various abiotic and biotic factors. Accumulation of ROS is damaging to various cellular components and macromolecules including plasma membrane, nucleic acids, and proteins and eventually leads to cell death. In this protocol, we describe the histochemical detection of hydrogen peroxide (H2O2) and superoxide (O2-) anion, two of the most important ROS, in Brassica juncea seedlings by using 3,3ʹ-Diaminobenzidine (DAB) and Nitrotetrazolium blue chloride (NBT) as the chromogenic substrate. DAB is oxidized by H2O2 in the presence of peroxidases and produces reddish brown precipitate. NBT reacts with O2- to form a dark blue insoluble formazan compound. The protocol can be used in other plant species and for different plant tissues., 植物细胞不断产生活性氧（ROS）作为有氧代谢的副产物。增加的ROS的产生发生在由各种非生物和生物因素施加的不利条件下。 ROS的积累损害各种细胞组分和大分子，包括质膜，核酸和蛋白质，并最终导致细胞死亡。在该方案中，我们描述了过氧化氢（H 2 O 2 O 2）和超氧化物（O 2 2 O 2）的组织化学检测，通过使用3,3'-二氨基联苯胺（DAB）和硝基四唑氯化蓝（NBT）作为显色底物，在芥菜籽苗中培养两种最重要的ROS。 DAB在过氧化物酶存在下被H 2 O 2 O 2氧化并产生红棕色沉淀。 NBT与O 2反应以形成深蓝色不溶性甲an化合物。该方案可用于其他植物物种和不同的植物组织。

Influence of Light on the Free Amino Acid Content and γ-Aminobutyric Acid Synthesis in Brassica juncea Seedlings

Glutamate decarboxylase (GAD; EC 4.1.1.15) is an important enzyme in γ-aminobutyric acid (GABA) biosynthesis. Here we report the influence of light on amino acid accumulation and investigate the molecular mechanism by which light influences GABA biosynthesis at the seedling stage of two mustard (Brassica juncea) cultivars (green-leaf and purple-leaf). Gene expression profiles of four GAD-encoding genes (GAD1, GAD2, GAD4a, and GAD4b) and their impact on GABA biosynthesis were analyzed. Light exerted an obvious influence on amino acid accumulation in mustard seedlings. GAD gene expression was also significantly regulated by light/dark or dark treatment, which differentially regulated GABA biosynthesis in B. juncea seedlings. High-performance liquid chromatography (HPLC) revealed that the seeds of purple cultivars contain a higher amount of free amino acids and GABA than do the seeds of green cultivars. After seed germination, however, the accumulation of free amino acids peaked in dark-treated seedlings on day 9 in both cultivars, whereas GABA synthesis peaked at 9 days under light conditions. This study may provide a foundation for understanding the effect of light on amino acids, particularly GABA biosynthesis in Brassica plants.

RuBisCO depletion improved proteome coverage of cold responsive S-nitrosylated targets in Brassica juncea

Although in the last few years good number of S-nitrosylated proteins are identified but information on endogenous targets is still limiting. Therefore, an attempt is made to decipher NO signaling in cold treated Brassica juncea seedlings. Treatment of seedlings with substrate, cofactor and inhibitor of Nitric-oxide synthase and nitrate reductase (NR), indicated NR mediated NO biosynthesis in cold. Analysis of the in vivo thiols showed depletion of low molecular weight thiols and enhancement of available protein thiols, suggesting redox changes. To have a detailed view, S-nitrosylation analysis was done using biotin switch technique (BST) and avidin-affinity chromatography. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is S-nitrosylated and therefore, is identified as target repeatedly due to its abundance. It also competes out low abundant proteins which are important NO signaling components. Therefore, RuBisCO was removed (over 80%) using immunoaffinity purification. Purified S-nitrosylated RuBisCO depleted proteins were resolved on 2-D gel as 110 spots, including 13 new, which were absent in the crude S-nitrosoproteome. These were identified by nLC-MS/MS as thioredoxin, fructose biphosphate aldolase class I, myrosinase, salt responsive proteins, peptidyl-prolyl cis-trans isomerase and malate dehydrogenase. Cold showed differential S-nitrosylation of 15 spots, enhanced superoxide dismutase activity (via S-nitrosylation) and promoted the detoxification of superoxide radicals. Increased S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase sedoheptulose-biphosphatase, and fructose biphosphate aldolase, indicated regulation of Calvin cycle by S-nitrosylation. The results showed that RuBisCO depletion improved proteome coverage and provided clues for NO signaling in cold.

Brassica juncea nitric oxide synthase like activity is stimulated by PKC activators and calcium suggesting modulation by PKC-like kinase

Nitric oxide (NO) is an important signaling molecule having varied physiological and regulatory roles in biological systems. The fact that nitric oxide synthase (NOS) is responsible for NO generation in animals, prompted major search for a similar enzyme in plants. Arginine dependent NOS like activity (BjNOSla) was detected in Brassica juncea seedlings using oxyhemoglobin and citrulline assays. BjNOSla showed 25% activation by NADPH (0.4mM) and 40% by calcium (0.4mM) but the activity was flavin mononucleotide (FMN), flavin dinucleotide (FAD) and calmodulin (CaM) independent. Pharmacological approach using mammalian NOS inhibitors, NBT (300μM) and l-NAME (5mM), showed significant inhibition (100% and 67% respectively) supporting that the BjNOSla operates via the oxidative pathway. Most of the BjNOSla activity (80%) was confined to shoot while root showed only 20% activity. Localization studies by NADPH-diaphorase and DAF-2DA staining showed the presence of BjNOSla in guard cells. Kinetic analysis showed positive cooperativity with calcium as reflected by a decreased K(m) (∼13%) and almost two fold increase in V(max). PMA (438nM), a kinase activator, activated BjNOSla ∼1.9 fold while its inactive analog 4αPDD was ineffective. Calcium and PMA activated the enzyme to ∼3 folds. Interestingly, 1,2-DG6 (2.5μM) and PS (1μM) with calcium activated the enzyme activity to ∼7 fold. A significant inhibition of BjNOSla by PKC inhibitors-staurosporine (∼90%) and calphostin-C (∼40%), further supports involvement of PKC-like kinase. The activity was also enhanced by abiotic stress conditions (7-46%). All these findings suggest that BjNOSla generates NO via oxidative pathway and is probably regulated by phosphorylation.

Silver Nanoparticle-Mediated Enhancement in Growth and Antioxidant Status of Brassica juncea

Metal nanoparticles can potentially be used as tools for engineering biological redox reactions. Present study underlines the effect of silver metal nanoparticles (at 0, 25, 50, 100, 200 and 400 ppm) on the growth and antioxidant status of 7-day-old Brassica juncea seedlings. Fresh weight, root and shoot length, and vigor index of seedlings is positively affected by silver nanoparticle treatment. It induced a 326 % increase in root length and 133 % increase in vigor index of the treated seedlings. Improved photosynthetic quantum efficiency and higher chlorophyll contents were recorded in leaves of treated seedlings, as compared to the control seedlings. Levels of malondialdehyde and hydrogen peroxide decreased in the treated seedlings. Nanoparticle treatment induced the activities of specific antioxidant enzymes, resulting in reduced reactive oxygen species levels. Decrease in proline content confirmed the improvement in antioxidant status of the treated seedlings. The observed stimulatory affects of silver nanoparticles are found to be dose dependent, with 50 ppm treatment being optimum for eliciting growth response. Present findings, for the first time indicate that silver nanoparticles promote the growth of B. juncea seedlings by modulating their antioxidant status.