Structural and functional analysis of Chitinase-IV of Brassica juncea: molecular modeling and dynamic simulation study.

Brassica juncea is an important oil seed crop. The productivity of this plant, however, is known to be low due to the attack of plant pathogens. The plant chitinase-IV is known to hydrolyse the chitin present in the cell walls of the plant pathogens and thus enhance the plant defense systems. In this connection, studies were carried out by us on the prediction and characterization of the 3D structure of chitinase-IV, the structural changes that take place when the protein is in complex with Allosamidin and the chitin fragments (Tri-oligosaccharide and N-acetyl glucosamine) that act as elicitors to induce plant innate immunity against the invading pathogens, and molecular dynamic simulation studies on the stability of these complexes. These studies are expected to give us an insight into the chitin-binding domain and information on the dynamics and energetics of the protein, which is not possible to obtain by experimental methods. The predicted 3D structure of the protein should give us a better understanding of the molecular function of the chitinase gene in Brassica juncea for devising better methods of biocontrol against fungal phytopathogens and harmful insects so as to increase the crop yield.

Overexpression of LYK4, a lysin motif receptor with non-functional kinase domain, enhances tolerance to Alternaria brassicicola and increases trichome density in Brassica juncea.

Lysin motif receptor-like kinases (LYKs) are involved in the recognition of chitin and activation of plant immune response. In this study, we found LYK4 to be strongly induced in resistant Sinapis alba compared with susceptible Brassica juncea on challenge with Alternaria brassicicola. In silico analysis and in vitro kinase assay revealed that despite the presence of canonical protein kinase fold, B.juncea LYK4 (BjLYK4) lacks several key residues of a prototype protein kinase which renders it catalytically inactive. Transient expression analysis confirmed that fluorescently tagged BjLYK4 localizes specifically to the plasma membrane. Overexpression (OE) of BjLYK4 in B. juncea enhanced tolerance against A. brassicicola. Interestingly, the OE lines also exhibited a novel trichome dense phenotype and increased jasmonic acid (JA) responsiveness. We further showed that many chitin responsive WRKY transcription factors and JA biosynthetic genes were strongly induced in the OE lines on challenge with the pathogen. Moreover, several JA inducible trichome developmental genes constituting the WD-repeat/bHLH/MYB activator complex were also upregulated in the OE lines compared with vector control and RNA interference line. These results suggest that BjLYK4 plays an essential role in chitin-dependent activation of defense response and chitin independent trichome development likely by influencing the JA signaling pathway.

[Mutant construction of HDA9 and its interactions with promoters of flowering integrator SOC1 and AGL24 in Brassica juncea].

HDA9, a member of the deacetylase family, plays a vital role in regulating plant flowering time through flowering integrator SOC1 and AGL24. However, it remains elusive how HDA9 interacts with SOC1 and AGL24 in flowering time control. Here, HDA9 was cloned in Brassica juncea and then its three active sites were separately replaced with Ala via overlap extension PCR. Thus, mutants of HDA9(D172A), HDA9(H174A) and HDA9(D261A) were constructed and fused into the pGADT7 vector. The yeast one-hybrid assays indicated that HDA9 mutants remained the interactions with the promoters of SOC1 and AGL24. Furthermore, the aforementioned results were confirmed in the dual luciferase assays. Interestingly, the DNA-protein interactions were weakened significantly due to the mutation in the three active sites of HDA9. It suggested that flowering signal integrator SOC1 and AGL24 were regulated by the key amino acid residues of 172th, 174th and 261th in HDA9. Our results provide valuable information for the in-depth study of the biological function and molecular regulation of HDA9 in Brassica juncea flowering time control.

Expression Analysis of XTH in Stem Swelling of Stem Mustard and Selection of Reference Genes.

Accurate analysis of gene expression requires selection of appropriate reference genes. In this study, we report analysis of eight candidate reference genes (ACTIN, UBQ, EF-1α, UBC, IF-4α, TUB, PP2A, and HIS), which were screened from the genome and transcriptome data in Brassica juncea. Four statistical analysis softwares geNorm, NormFinder, BestKeeper, and RefFinder were used to test the reliability and stability of gene expression of the reference genes. To further validate the stability of reference genes, the expression levels of two CYCD3 genes (BjuB045330 and BjuA003219) were studied. In addition, all genes in the xyloglucan endotransglucosylase/hydrolase (XTH) family were identified in B. juncea and their patterns at different periods of stem enlargement were analyzed. Results indicated that UBC and TUB genes showed stable levels of expression and are recommended for future research. In addition, XTH genes were involved in regulation of stem enlargement expression. These results provide new insights for future research aiming at exploring important functional genes, their expression patterns and regulatory mechanisms for mustard development.

Genome-wide identification and characterization of Chitinase gene family in Brassica juncea and Camelina sativa in response to Alternaria brassicae.

Chitinases belong to the group of Pathogenesis-related (PR) proteins that provides protection against fungal pathogens. This study presents the, genome-wide identification and characterization of chitinase gene family in two important oilseed crops B. juncea and C. sativa belonging to family Brassicaceae. We have identified 47 and 79 chitinase genes in the genomes of B. juncea and C. sativa, respectively. Phylogenetic analysis of chitinases in both the species revealed four distinct sub-groups, representing different classes of chitinases (I-V). Microscopic and biochemical study reveals the role of reactive oxygen species (ROS) scavenging enzymes in disease resistance of B. juncea and C. sativa. Furthermore, qRT-PCR analysis showed that expression of chitinases in both B. juncea and C. sativa was significantly induced after Alternaria brassicae infection. However, the fold change in chitinase gene expression was considerably higher in C. sativa compared to B. juncea, which further proves their role in C. sativa disease resistance to A. brassicae. This study provides comprehensive analysis on chitinase gene family in B. juncea and C. sativa and in future may serve as a potential candidate for improving disease resistance in B. juncea through transgenic approach.

Identification and functional characterization of a novel selenocysteine methyltransferase from Brassica juncea L.

Organic selenium (Se), specifically Se-methylselenocysteine (MeSeCys), has demonstrated potential effects in human disease prevention including cancer and the emerging ameliorating effect on Alzheimer's disease. In plants, selenocysteine methyltransferase (SMT) is the key enzyme responsible for MeSeCys formation. In this study, we first isolated a novel SMT gene, designated as BjSMT, from the genome of a known Se accumulator, Brassica juncea L. BjSMT shows high sequence (amino acid) similarity with its orthologues from Brassica napus and Brassica oleracea var. oleracea, which can use homocysteine (HoCys) and selenocysteine (SeCys) as substrates. Similar to its closest homologues, BjSMT also possesses a conserved Thr187 which is involved in transferring a methyl group to HoCys by donating a hydrogen bond, suggesting that BjSMT can methylate both HoCys and SeCys substrates. Using quantitative real-time PCR (qRT-PCR) technology and BjSMT-transformed tobacco (Nicotiana tabacum) plants, we observed how BjSMT responds to selenite [Se(IV)] and selenate [Se(VI)] stress in B. juncea, and how the phenotypes of BjSMT-overexpressing tobacco cultured under selenite stress are affected. BjSMT expression was nearly undetectable in the B. juncea plant without Se exposure, but in the plant leaves it can be rapidly and significantly up-regulated upon a low level of selenite stress, and enormously up-regulated upon selenate treatment. Overexpression of BjSMT in tobacco substantially enhanced tolerance to selenite stress manifested as significantly higher fresh weight, plant height, and chlorophyll content than control plants. In addition, transgenic plants exhibited low glutathione peroxidase activity in response to a lower dose of selenite stress (with a higher dose of selenite stress resulting in a high activity response) compared with the controls. Importantly, the BjSMT-transformed tobacco plants accumulated a high level of Se upon selenite stress, and the plants also had significantly increased MeSeCys production potential in their leaves. This first study of B. juncea SMT demonstrates its potential applications in crop MeSeCys biofortification and phytoremediation of Se pollution.

High-efficiency antioxidant system, chelating system and stress-responsive genes enhance tolerance to cesium ionotoxicity in Indian mustard (Brassica juncea L.).

Indian mustard (Brassica juncea L.) was more tolerance to Cs than some sensitive plants, such as Arabidopsis thaliana and Vicia faba, and may have a special detoxification mechanism. In this study, the effects on reactive oxygen species (ROS) content, the antioxidant enzyme system and chelation system in Indian mustard were studied by observing different plant physiological responses. In addition, we focused on the analysis of gene regulatory networks related to ROS formation, ROS scavenging system, and other stress-response genes to Cs exposure using a transcriptome-sequencing database. The results showed that ROS and malonaldehyde content in seedlings increased significantly in Cs-treatment groups. The enzyme activities of superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase were increased, and the synthesis of antioxidants glutathione, phytochelatin and metallothionein also increased under Cs treatment. Further analysis showed that ROS formation pathways were primarily the photosynthetic electron transport chain process and photorespiration process in the peroxisome. Antioxidant enzyme systems and the respiratory burst oxidase homolog protein-mediated signal transduction pathway played a key role in ROS scavenging. In summary, one of the mechanisms of tolerance and detoxification of Indian mustard to Cs was that it enhanced the scavenging ability of antioxidant enzymes to ROS, chelated free Cs ions in cells and regulated the expression of related disease-resistant genes.

Streptomyces pactum and sulfur mediated the antioxidant enzymes in plant and phytoextraction of potentially toxic elements from a smelter-contaminated soils.

The toxic potentially toxic metals elements (PTEs) discharged from industrial activities and agricultural practices persistently pose multiple hazards to environment and living organisms. Microbe-assisted phytoremediation provide an effective approach to remediate PTEs-contaminated soils. A phytoextraction process involved the application of Streptomyces pactum (Act12, 1.0, 2.0 and 3.0 g kg-1 dry soil, respectively) alone/jointly with sulfur was executed. The main texture of the tested soil was sandy loam and with a pH 8.27. The obtained results showed that the leaf pigments and plant biomass were improved after the application of the Act12, while the shoot fresh weight, chlorophyll a and chlorophyll b decreased by 57.8, 38.2 and 40.7%, respectively, after treatment with sulfur. Similarly, sulfur application facilitated the malondialdehyde (MDA) production by 18.4-33.6% compared to the control (no amendments). Both peroxidase (POD) and superoxide dismutase (SOD) activities were boosted, while the catalase (CAT) activity was suppressed with Act12 alone/jointly with sulfur treatment. The sulfur combined with elevated Act12 levels notably increased the cadmium (Cd) and zinc (Zn) concentrations both in shoots and roots, while the elemental extraction amount showed the removal efficiency following the order: Act12 alone ＞ control ＞ Act12 jointly with sulfur. Taken together, the results suggested that Streptomyces pactum and sulfur assisted the phytoremediation process, while further studies should be conducted in the field to test practical application.

Genetic analyses of nitrogen assimilation enzymes in Brassica juncea (L.) Czern & Coss.

Nitrogen (N) is a critical input for plant growth and development. A better understanding of N uptake and utilization is important to develop plant breeding strategies for improving nitrogen use efficiency (NUE). With that objective in mind, we assayed a SNP-genotyped association panel comprising 92 inbred lines for the activities of nitrate reductase (NR), nitrite reductase (NIR), glutamine synthetase (GS) and glutamate synthase (GOGAT). All these enzymes are associated with N assimilation. The experiments were carried out at two levels of N application: no added N (N0) and agrnomically recommened dose (100 kg/ha) of N application (N100). Genome wide association studies (GWAS) helped to identify several marker-trait associations (MTAs), involving chromosomes A01, A06, A08, B02, B04, B05 and B08. These explained high phenotypic variation (up to 32%). Annotation of the genomic region(s) in or around significant SNPs allowed prediction of genes encoding high affinity nitrate transporters, glutamine synthetase 1.3, myb-like transcription factor family protein, bidirectional amino acid transporter 1, auxin signaling F-box 3 and oxidoreductases. This is the first attempt to use GWAS for identification of enzyme QTLs to explain variation for nitrogen assimilation enzymes in Brassica juncea.

Salicylic acid enhances nickel stress tolerance by up-regulating antioxidant defense and glyoxalase systems in mustard plants.

The present study identified inverse relationships between nickel (Ni) levels and growth, photosynthesis and physio-biochemical attributes, but increasing levels of Ni stress enhanced methylglyoxal, electrolyte leakage, hydrogen peroxide, and lipid peroxidation content. Exogenous application of salicylic acid (SA) (10-5 M) ameliorated the ill-effects of Ni by restoring growth, photosynthesis and physio-biochemical attributes and increasing the activities of enzymes associated with antioxidant systems, especially the ascorbate-glutathione (AsA-GSH) cycle and glyoxalase system. In addition, SA application to Ni-stressed plants had an additive effect on the activities of the ascorbate and glutathione pools, and the AsA-GSH cycle enzymes (ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase), superoxide dismutase, catalase, glutathione S-transferase, and osmolyte biosynthesis). This trend also follows in glyoxalase system viz. glyoxalase I and glyoxalase II enzymes. Nevertheless, exogenous SA supplementation restored mineral nutrient contents. Principal component analysis showed that growth, photosynthesis, and mineral nutrient parameters were positively correlated with each other and negatively correlated with antioxidant enzymes and oxidative stress biomarkers. Hence, SA is an alternative compound with potential application in the phytoremediation of Ni.

Myrosinase: insights on structural, catalytic, regulatory, and environmental interactions.

Glucosinolate-myrosinase is a substrate-enzyme defense mechanism present in Brassica crops. This binary system provides the plant with an efficient system against herbivores and pathogens. For humans, it is well known for its anti-carcinogenic, anti-inflammatory, immunomodulatory, anti-bacterial, cardio-protective, and central nervous system protective activities. Glucosinolate and myrosinase are spatially present in different cells that upon tissue disruption come together and result in the formation of a variety of hydrolysis products with diverse physicochemical and biological properties. The myrosinase-catalyzed reaction starts with cleavage of the thioglucosidic linkage resulting in release of a D-glucose and an unstable thiohydroximate-O-sulfate. The outcome of this thiohydroximate-O-sulfate has been shown to depend on the structure of the glucosinolate side chain, the presence of supplementary proteins known as specifier proteins and/or on the physiochemical condition. Myrosinase was first reported in mustard seed during 1939 as a protein responsible for release of essential oil. Until this date, myrosinases have been characterized from more than 20 species of Brassica, cabbage aphid, and many bacteria residing in the human intestine. All the plant myrosinases are reported to be activated by ascorbic acid while aphid and bacterial myrosinases are found to be either neutral or inhibited. Myrosinase catalyzes hydrolysis of the S-glycosyl bond, O-ß glycosyl bond, and O-glycosyl bond. This review summarizes information on myrosinase, an essential component of this binary system, including its structural and molecular properties, mechanism of action, and its regulation and will be beneficial for the research going on the understanding and betterment of the glucosinolate-myrosinase system from an ecological and nutraceutical perspective.

Genome-wide identification and characterization of abiotic-stress responsive SOD (superoxide dismutase) gene family in Brassica juncea and B. rapa.

BACKGROUND: Abiotic stresses like drought, heat, cold and salinity cause major productivity loss in the rapeseed-mustard crops (Brassica). Major efforts have been made in the past to identify genes that provide resistance against such stresses. Superoxide dismutase (SOD) proteins, member of the metallo-enzyme family play vital role in protecting plants against abiotic stresses. In the present study, genome-wide analysis of abiotic stress responsive SOD gene family has been done in B. juncea and B. rapa. RESULTS: A total of 29 and 18 SOD genes were identified in B. juncea and B. rapa respectively and chromosome location mapping indicated their wide distribution across genome. On the basis of domain composition, the SODs were phylogenetically classified into sub-groups which was also substantiated by the gene structure and sub-cellular locations of SOD proteins. Functional annotation of SODs was also done by Gene Ontology (GO) mapping and the result was corroborated by the identified cis-regulatory elements in the promoter region of SOD genes. Based on FPKM analysis of SRA data available for drought, heat and salt stress, we identified 14 and 10 abiotic stress responsive SOD genes in B. rapa and B. juncea respectively. The differential expression analysis under drought and heat stress of identified abiotic-stress responsive SOD genes was done through quantitative Real Time PCR. CONCLUSION: We identified abiotic-stress responsive genes that could help in improving the plant tolerance against abiotic stresses. This was the first study to describe the genome-wide analysis of SOD gene family in B. rapa and B. juncea, and the results will help in laying basic ground for future work of cloning and functional validation of SOD genes during abiotic stresses leading to Brassica crop improvement.

28-homobrassinolide regulates antioxidant enzyme activities and gene expression in response to salt- and temperature-induced oxidative stress in Brassica juncea.

Brassinosteroids (BRs) are a group of naturally occurring plant steroid hormones that can induce plant tolerance to various plant stresses by regulating ROS production in cells, but the underlying mechanisms of this scavenging activity by BRs are not well understood. This study investigated the effects of 28-homobrassinolide (28-HBL) seed priming on Brassica juncea seedlings subjected to the combined stress of extreme temperatures (low, 4 °C or high, 44 °C) and salinity (180 mM), either alone or supplemented with 28-HBL treatments (0, 10-6, 10-9, 10-12 M). The combined temperature and salt stress treatments significantly reduced shoot and root lengths, but these improved when supplemented with 28-HBL although the response was dose-dependent. The combined stress alone significantly increased H2O2 content, but was inhibited when supplemented with 28-HBL. The activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APOX), glutathione reductase (GR), dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) increased in response to 28-HBL. Overall, the 28-HBL seed priming treatment improved the plant's potential to combat the toxic effects imposed by the combined temperature and salt stress by tightly regulating the accumulation of ROS, which was reflected in the improved redox state of antioxidants.

Simultaneous exposure of sulphur and calcium hinder As toxicity: Up-regulation of growth, mineral nutrients uptake and antioxidants system.

The current study was carried out to investigate the role of exogenous sulphur (K2SO4: S; 60 mg S kg-1 sand) and calcium (CaCl2: Ca; 250 mg Ca kg-1 sand) individually as well as in combination (S + Ca) in ameliorating the inhibitory effect of As (Na2HAsO4·7H2O: As1; 15 mg As kg-1 sand and As2; 30 mg As kg-1 sand) by analyzing biomass accumulation, mineral nutrients uptake, photosynthetic pigments content, redox status of the cell, enzymatic and non-enzymatic defense system in Brassica juncea L. seedlings. Biomass accumulation, uptake of mineral nutrients, photosynthetic pigments (chlorophyll a, b and carotenoids) content and the activity of proline dehydrogenase (ProDH) declined with increasing accumulation of As in root as well as leaves in As dose dependent manner. Contrary to this, exogenous application of S, Ca and S + Ca, markedly reduced the negative impact of As on above captioned traits except ProDH activity. On the other hand, ROS and their biomarkers (superoxide radical; O2˙-, hydrogen peroxide; H2O2, malondialdehyde; MDA equivalents content and membrane damage; electrolyte leakage), activities of enzymatic (superoxide dismutase; SOD, peroxidase; POD, catalase; CAT and glutathione-S-transferase; GST) and non-enzymatic antioxidant i.e. proline (Pro) content and its enzyme pyrroline-5-carboxylate synthetase; P5CS activity were increased in root and leaves under As stress. While, exogenous application of S, Ca and S + Ca, further enhanced the activities of above mentioned enzymes and Pro content thereby causing considerable reduction in O2˙-, H2O2, MDA equivalents content and electrolyte leakage. This study suggests that exogenous application of S and/or Ca efficiently (particularly S + Ca) lowered the negative impact of As on biomass accumulation in Brassica seedlings by improving the uptake of essential mineral nutrients', content of photosynthetic pigments, activities of enzymatic and content of non-enzymatic antioxidants.

Supplementation of the Diet by Exogenous Myrosinase via Mustard Seeds to Increase the Bioavailability of Sulforaphane in Healthy Human Subjects after the Consumption of Cooked Broccoli.

SCOPE: Broccoli contains glucosinolate glucoraphanin, which, in the presence of myrosinase, can hydrolyze to isothiocyanate sulforaphane, reported to have anticarcinogenic activity. However, the myrosinase enzyme is denatured on cooking. Addition of an active source of myrosinase, such as from powdered mustard seed, to cooked Brassica vegetables can increase the release of health beneficial isothiocyanates; however, this has not previously been proven in vivo. METHODS AND RESULTS: The concentration of sulforaphane metabolite (sulforaphane N-acetyl-l-cysteine [SF-NAC]) in 12 healthy adults after the consumption of 200 g cooked broccoli, with and without 1 g powdered brown mustard, was studied in a randomized crossover design. During the 24-h period following the consumption of the study sample, all urine was collected. SF-NAC content was assayed by HPLC. When study subjects ingested cooked broccoli alone, mean urinary SF-NAC excreted was 9.8 ± 5.1 µmol per g creatinine, and when cooked broccoli was consumed with mustard powder, this increased significantly to 44.7 ± 33.9 µmol SF-NAC per gram creatinine. CONCLUSION: These results conclude that when powdered brown mustard is added to cooked broccoli, the bioavailability of sulforaphane is over four times greater than that from cooked broccoli ingested alone.

Interaction of 24-epibrassinolide and salicylic acid regulates pigment contents, antioxidative defense responses, and gene expression in Brassica juncea L. seedlings under Pb stress.

Lead (Pb) is considered one the most hazardous pollutant, and its accumulation in soil and plants is of prime concern. To understand the role of plant hormones in combating heavy metal stress, the present study was planned to assess the interactive effects of 24-epibrassinolide (EBL) (10-7 M) and salicylic acid (SA) (1 mM) in regulating growth, pigment contents, antioxidative defense response, and gene expression in Brassica juncea L. seedlings exposed to different concentrations of Pb metal (0.25, 0.50, and 0.75 mM). Reduction in root and shoot lengths, chlorophyll and carotenoid content, and non-enzymatic antioxidants like glutathione, ascorbic acid, and tocopherol in response to Pb toxicity was observed. The enzymatic antioxidants such as guaiacol peroxidase (POD), ascorbate peroxidase (APOX), glutathione peroxidase (GR), dehydroascorbate reductase (DHAR), monodehydroascorbate redductase (MDHAR), glutathione-S-transferease (GST), and glutathione peroxidase (GPOX) were lowered in response to Pb treatments. Other antioxidative enzymes including superoxide dismutase (SOD), catalase (CAT), and polyphenol oxidase (PPO) enhanced under metal stress. Co-application of EBL + SA to 0.75 mM Pb-treated seedlings resulted in improvement of root and shoot lengths, chlorophyll, and carotenoid contents. Similarly, glutathione, ascorbic acid, and tocopherol contents were also elevated. Enzymatic antioxidants were also significantly enhanced in response to pre-sowing combined treatment of both hormones. Gene expression analysis suggested elevation in expression of CAT, POD, GR, DHAR, and GST genes by application of EBL. Our results reveal that Pb metal toxicity caused adverse impact on B. juncea L. seedlings, but pre-soaking treatment with EBL and SA individually and in combination help seedlings to counter the ill effects of Pb by improving growth, contents of pigment, and modulation of antioxidative defense system. The combined application of EBL and SA was found to be more effective in ameliorating Pb stress as compared to their individual treatments.

Modulation of antioxidative defense expression and osmolyte content by co-application of 24-epibrassinolide and salicylic acid in Pb exposed Indian mustard plants.

The study focuses on potential of combined pre-soaking treatment of 24-Epibrassinolide (EBL) and Salicylic acid (SA) in alleviating Pb phytotoxicity in Brassica juncea L. plants. The seeds after treatment with combination of both the hormones were sown in mixture of soil, sand and manure (3:1:1) and were exposed to Pb concentrations (0.25mM, 0.50mM and 0.75mM). After 30 days of growth, the plants were harvested and processed, for quantification of various metabolites. It was found that pre-sowing of seeds in combination of EBL and SA, mitigated the adverse effects of metal stress by modulating antioxidative defense response and enhanced osmolyte contents. Dry matter content and heavy metal tolerance index were enhanced in response to co-application of EBL and SA. The levels of superoxide anions, hydrogen peroxide and malondialdehyde were lowered by the combined treatment of hormones. Enhancement in activities of guaiacol peroxidase, catalase, glutathione reductase and glutathione-s-transferase were recorded. Contents of glutathione, tocopherol and ascorbic acid were also enhanced in response to co-application of both hormones. Expression of POD, CAT, GR and GST1 genes were up-regulated whereas SOD gene was observed to be down-regulated. Contents of proline, trehalose and glycine betaine were also reported to be elevated as a result of treatment with EBL+SA. The results suggest that co-application of EBL+SA may play an imperative role in improving the antioxidative defense expression of B. juncea plants to combat the oxidative stress generated by Pb toxicity.

Improved fruit α-tocopherol, carotenoid, squalene and phytosterol contents through manipulation of Brassica juncea 3-HYDROXY-3-METHYLGLUTARYL-COA SYNTHASE1 in transgenic tomato.

3-Hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) in the mevalonate (MVA) pathway generates isoprenoids including phytosterols. Dietary phytosterols are important because they can lower blood cholesterol levels. Previously, the overexpression of Brassica juncea wild-type (wt) and mutant (S359A) BjHMGS1 in Arabidopsis up-regulated several genes in sterol biosynthesis and increased sterol content. Recombinant S359A had earlier displayed a 10-fold higher in vitro enzyme activity. Furthermore, tobacco HMGS overexpressors (OEs) exhibited improved sterol content, plant growth and seed yield. Increased growth and seed yield in tobacco OE-S359A over OE-wtBjHMGS1 coincided with elevations in NtSQS expression and sterol content. Herein, the overexpression of wt and mutant (S359A) BjHMGS1 in a crop plant, tomato (Solanum lycopersicum), caused an accumulation of MVA-derived squalene and phytosterols, as well as methylerythritol phosphate (MEP)-derived α-tocopherol (vitamin E) and carotenoids, which are important to human health as antioxidants. In tomato HMGS-OE seedlings, genes associated with the biosyntheses of C10, C15 and C20 universal precursors of isoprenoids, phytosterols, brassinosteroids, dolichols, methylerythritol phosphate, carotenoid and vitamin E were up-regulated. In OE-S359A tomato fruits, increased squalene and phytosterol contents over OE-wtBjHMGS1 were attributed to heightened SlHMGR2, SlFPS1, SlSQS and SlCYP710A11 expression. In both tomato OE-wtBjHMGS1 and OE-S359A fruits, the up-regulation of SlGPS and SlGGPPS1 in the MEP pathway that led to α-tocopherol and carotenoid accumulation indicated cross-talk between the MVA and MEP pathways. Taken together, the manipulation of BjHMGS1 represents a promising strategy to simultaneously elevate health-promoting squalene, phytosterols, α-tocopherol and carotenoids in tomato, an edible fruit.

Castasterone confers copper stress tolerance by regulating antioxidant enzyme responses, antioxidants, and amino acid balance in B. juncea seedlings.

The aim of the present study was to explore the effect of exogenous application of castasterone (CS) on physiologic and biochemical responses in Brassica juncea seedlings under copper (Cu) stress. Seeds were pre-soaked in different concentrations of CS and grown for 7 days under various levels of Cu. The exposure of B. juncea to higher levels of Cu led to decrease of morphologic parameters, with partial recovery of length and fresh weight in the CS pre-treated seedlings. Metal content was high in both roots and shoots under Cu exposure while the CS pre-treatment reduced the metal uptake. Accumulation of hydrogen peroxide (H2O2) and superoxide anion radical (O2-) were chosen as stress biomarker and higher levels of H2O2 (88.89%) and O2- (62.11%) showed the oxidative stress in metal treated B. juncea seedlings, however, CS pre-treatment reduced ROS accumulation in Cu-exposed seedlings. The Cu exposures lead to enhance the plant's enzymatic and non-enzymatic antioxidant system. It was observed that enzymatic activities of ascorbate peroxidase (APOX), dehydroascorbate reductase (DHAR), and glutathione reductase (GR), glutathione perxoidase (GPOX) and gultrathione-s-transferase increased while activity of monodehydroascorbate reductase (MDHAR) decreased under Cu stress. The pre-treatment with CS positively affected the activities of enzymes. RT-PCR analysis showed that mRNA transcript levels were correlated with total enzymatic activity of DHAR, GR, GST and GSH. Increase in the gene expression of DHAR (1.85 folds), GR (3.24 folds), GST-1 (2.00 folds) and GSH-S (3.18 folds) was noticed with CS pre-treatment. Overall, the present study shows that Cu exposure induced severe oxidative stress in B. juncea plants and exogenous application of CS improved antioxidative defense system by modulating the ascorbate-glutathione cycle and amino acid metabolism.

Castasterone and citric acid treatment restores photosynthetic attributes in Brassica juncea L. under Cd(II) toxicity.

Cadmium(II) toxicity is a serious environmental issue warranting effective measures for its mitigation. In the present study, ameliorative effects of a bioactive brassinosteroid, castasterone (CS) and low molecular weight organic acid, citric acid (CA) against the Cd(II) toxicity to Brassica juncea L. were evaluated. Seeds of B. juncea treated with CS (0, 0.01, 1 and 100nM) were sown in cadmium spiked soils (0 and 0.6mmolkg-1 soil). CA (0.6mmolkg-1soil) was added to soil one week after sowing seeds. Plants were harvested 30 days after sowing. Phytotoxicity induced by Cd(II) was evident from stunted growth of the plants, malondialdehyde accumulation, reduction in chlorophyll and carotenoid contents, and leaf gas exchange parameters. Cd(II) toxicity was effectively alleviated by seed soaking with CS (100nM) and/ or soil amendment with CA (0.6mMkg-1 soil). Relative gene expression of genes encoding for some of the key enzymes of pigment metabolism were also analysed. Expression of chlorophyllase (CHLASE) was reduced, while that of phytoene synthase (PSY), and chalcone synthase (CHS) genes were enhanced with CS and/or CA treatments with respect to plants treated with Cd(II) only. Cd also affected the activities of antioxidative enzymes. Plants responded to Cd(II) by accumulation of total sugars. CS (100nM) and CA treatments further enhanced the activities of these parameters and induced the contents of secondary plant pigments (flavonoids and anthocyanins) and proline. The results imply that seed treatment with CS and soil application with CA can effectively alleviate Cd(II) induced toxicity in B. juncea by strengthening its antioxidative defence system and enhancing compatible solute accumulation.