Exogenously applied melatonin enhanced the tolerance of Brassica napus against cobalt toxicity by modulating antioxidant defense, osmotic adjustment, and expression of stress response genes.

The excessive accumulation of cobalt (Co) in plant tissues severely impairs plant growth that ultimately reduces the yield. However, melatonin (MT) has been known to mediate the abiotic stress tolerance in plants. The present study aimed at investigating the protective mechanisms of exogenously applied MT (0, 50 and 100 µM) under Co (0, 100, 200 and 300 µM) stress by focusing on morpho-physiological, biochemical and cellular characterizations of Brassica napus plants. Cobalt (300 µM) alone treatment drastically inhibited the stomatal conductance, plant height (45%), leaf area (30%), free amino acid (139%), relative electrolyte leakage (109%), and total soluble sugars (71%), compared with the control. However, the exogenous supply of MT notably minimized the oxidative damage, lipid peroxidation and maintained the membrane integrity under Co-toxicity by restricting the overproduction of ROS (H2O2 and O2•), and MDA in leaves and roots. Melatonin significantly enhanced the activities of ROS-scavenging antioxidant enzymes, secondary metabolism-related phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), stress-responsive genes (heat shock protein as HSP-90, methyl transferase as MT) and regulated the Co-transporters, especially in roots. These findings indicated that an exogenous supply of MT improve the plant morphology, photosynthetic apparatus, osmotic adjustments, and antioxidant defense systems by enhancing the Co-detoxification in B. napus plants.

A High-Density Genetic Map of an Allohexaploid Brassica Doubled Haploid Population Reveals Quantitative Trait Loci for Pollen Viability and Fertility.

A doubled haploid (DH) mapping population was obtained from microspore culture of an allohexaploid F1 from the cross between two recently-synthesized allohexaploid Brassica lines. We used single nucleotide polymorphism (SNP) genetic variation based on restriction-site associated DNA (RAD) sequencing to construct a high density genetic linkage map of the population. RAD libraries were constructed from the genomic DNA of both parents and 146 DH progenies. A total of 2.87 G reads with an average sequencing depth of 2.59 × were obtained in the parents and of 1.41 × in the progeny. A total of 290,422 SNPs were identified from clustering of RAD reads, from which we developed 7,950 high quality SNP markers that segregated normally (1:1) in the population. The linkage map contained all 27 chromosomes from the parental A, B and C genomes with a total genetic distance of 5725.19 cM and an average of 0.75 cM between adjacent markers. Genetic distance on non-integrated linkage groups was 1534.23 cM, or 21% of total genetic distance. Out of 146 DH progenies, 91 had a complete set of 27 chromosomes as expected of a hexaploid species, and 21 out of 27 chromosomes showed high collinearity between the physical and linkage maps. The loss of chromosome(s) or chromosome segment(s) in the DH population was associated with a reduction in pollen viability. Twenty-five additive QTL were associated with pollen viability and fertility-related traits (seed number, seed yield, pod length, plant height, 1000-seed weight). In addition, 44 intra-genomic and 18 inter-genomic epistatic QTL pairs were detected for 4 phenotypic traits. This provides confidence that the DH population may be selected for improved pollen viability and fertility in a future allohexaploid Brassica species.

Ecotoxicological and Interactive Effects of Copper and Chromium on Physiochemical, Ultrastructural, and Molecular Profiling in Brassica napus L.

Heavy metal accumulation causes huge environmental problems, particularly in agricultural ecosystems which have deteriorative effects on the yield and quality of crops. Individual copper (Cu) and chromium (Cr) effects have been investigated extensively in plants; however, co-contamination of Cu and Cr induced stress on Brassica napus L. is still unclear. In the present experiment, the interactive effects of Cu and Cr were studied in two B. napus cultivars (Zheda 622 and ZS 758). Results showed that the application of Cr was more toxic than Cu, and their combined stress had shown a significant adverse effect on plant growth. Biomass and photosynthetic pigment were decreased remarkably under all metal treatments. Individual treatments of Cu and Cr and their combination cause the accumulation of ROS and lipid peroxidation. Moreover, the activities of antioxidant enzymes and their mRNA transcription levels, such as catalase (CAT), ascorbate peroxidase, glutathione reductase, superoxide dismutase, and peroxidase, were increased, especially when treated with Cr alone or under Cu+Cr combined treatment in both cultivars, except for the CAT activity which was decreased in both leaves and roots of sensitive cultivar Zheda 622 as compared with their respective controls. Additionally, nonenzymatic antioxidants like reduced and oxidized glutathione showed a differential activity pattern in roots and leaves of both cultivars. A more pronounced modification in chloroplast ultrastructure was observed in both cultivars under Cu+Cr treatment followed by Cr and Cu alone treatments. Furthermore, synergistic effects of Cu and Cr were prominent; this may be due to the enhanced metals uptake under combined treatment, which suggests that Cr and Cu interaction is not competitive but is rather additive and genotypic-dependent.

Comparative orchestrating response of four oilseed rape (Brassica napus) cultivars against the selenium stress as revealed by physio-chemical, ultrastructural and molecular profiling.

Selenium (Se) is an essential micro-element for human and animals. In higher plants, Se essentiality or phyto-toxicity is less explored. Therefore, we aimed to examine the effects of Se (0, 25, 50, and 100 µM) as sodium selenite on the physio-chemical, cell ultra-structural and genomic alterations in hydroponically grown seedlings of four cultivars of B. napus (cvs. Zheda 619, Zheda 622, ZS 758, and ZY 50). Results showed that excessive (100 µM) Se (IV) exhibited significant reduction in plant growth parameters, declined pigment contents, lower water-soluble protein levels, and overproduction of H2O2 and MDA contents. A significant increase in antioxidant enzyme activities and transcript levels of superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and glutathione reductase (GR), except catalase (CAT) were noticed in the leaves and roots. Non-enzymatic antioxidants including glutathione (GSH) and oxidized glutathione (GSSG), except GSSG in roots were enhanced under higher Se (IV) levels. Transmission electron microscopy analysis revealed the ultrastructural damages in leaf mesophyll and root tip cells induced by excessive Se (IV). Less-significant phytotoxic effects were observed in above-mentioned parameters at 50 µM Se (IV). Overall, Se (IV) supplementation at 25 µM displayed marginal beneficial effect by enhancing plant growth, pigment contents, protein levels and restrict H2O2 and MDA overproduction. A marginal increase/decrease in ROS-detoxifying enzymes (except CAT activity) and elevated GSH and GSSG levels were noticed. The accumulation of Se (IV) was much higher in roots as compared to leaves. This accumulation was maximum in Zheda 622 and minimum in ZS 758, followed by Zheda 619 and ZY 50. Overall findings showed that Zheda 622 was the most sensitive and ZS 758 as most tolerant to Se (IV) phyto-toxicity. In addition, Se (IV) was found beneficial until 25 µM Se (IV) but phytotoxic at higher Se levels especially at 100 µM Se (IV).

Beryllium Stress-Induced Modifications in Antioxidant Machinery and Plant Ultrastructure in the Seedlings of Black and Yellow Seeded Oilseed Rape.

Beryllium (Be) could be a threatening heavy metal pollutant in the agroecosystem that may severely affect the performance of crops. The present study was conducted to evaluate the toxic effects of Be (0, 100, 200, and 400 µM) on physiological, ultrastructure, and biochemical attributes in hydroponically grown six-day-old seedlings of two cultivars of Brassica napus L., one tolerant (ZS 758, black seeded) and one sensitive (Zheda 622, yellow seeded). Higher Be concentrations reduced the plant growth, biomass production, chlorophyll contents, and the total soluble protein contents. A significant accumulation of ROS (H2O2, OH-) and MDA contents was observed in a dose-dependent manner. Antioxidant enzymatic activities including SOD, POD, GR, APX, and GSH (except CAT) were enhanced with the increase in Be concentrations in both cultivars. Relative transcript gene expression of above-mentioned antioxidant enzymes further confirmed the alterations induced by Be as depicted from higher involvement in the least susceptible cultivar ZS 758 as compared to Zheda 622. The electron microscopic study showed that higher level of Be (400 µM) greatly damaged the leaf mesophyll and root tip cells. More damage was observed in cultivar Zheda 622 as compared to ZS 758. The damage in leaf mesophyll cells was highlighted as the disruption in cell wall, immature nucleus, damaged mitochondria, and chloroplast structures. In root tip cells, disruption in Golgi bodies and damage in cell wall were clearly noticed. As a whole, the present study confirmed that more inhibitory effects were recorded in yellow seeded Zheda 622 as compared to black seeded ZS 758 cultivar, which is regarded as more sensitive cultivar.

Salinity reduces 2,4-D efficacy in Echinochloa crusgalli by affecting redox balance, nutrient acquisition, and hormonal regulation.

Distinct salinity levels have been reported to enhance plants tolerance to different types of stresses. The aim of this research is to assess the interaction of saline stress and the use of 2,4-D as a means of controlling the growth of Echinochloa crusgalli. The resultant effect of such interaction is vital for a sustainable approach of weed management and food production. The results showed that 2,4-D alone treatment reduces the chlorophyll contents, photosynthetic capacity, enhanced MDA, electrolyte leakage, and ROS production (H2O2, O2·-) and inhibited the activities of ROS scavenging enzymes. Further analysis of the ultrastructure of chloroplasts indicated that 2,4-D induced severe damage to the ultrastructure of chloroplasts and thylakoids. Severe saline stress (8 dS m-1) followed by mild saline stress treatments (4 dS m-1) also reduced the E. crusgalli growth, but had the least impact as compared to the 2,4-D alone treatment. Surprisingly, under combined treatments (salinity + 2,4-D), the phytotoxic effect of 2,4-D was reduced on saline-stressed E. crusgalli plants, especially under mild saline + 2,4-D treatment. This stimulated growth of E. crusgalli is related to the higher activities of enzymatic and non-enzymatic antioxidants and dynamic regulation of IAA, ABA under mild saline + 2,4-D treatment. This shows that 2,4-D efficacy was affected by salinity in a stress intensity-dependent manner, which may result in the need for greater herbicide application rates, additional application times, or more weed control operations required for controlling salt-affected weed.

2,4-D attenuates salinity-induced toxicity by mediating anatomical changes, antioxidant capacity and cation transporters in the roots of rice cultivars.

Growth regulator herbicides are widely used in paddy fields to control weeds, however their role in conferring environmental stress tolerance in the crop plants are still elusive. In this study, the effects of recommended dose of 2,4-dichlorophenoxyacetic acid (2,4-D)  on growth, oxidative damage, antioxidant defense, regulation of cation transporter genes and anatomical changes in the roots of rice cultivars XS 134 (salt resistant) and ZJ 88 (salt sensitive) were investigated under different levels of saline stress. Individual treatments of saline stress and 2,4-D application induced oxidative damage as evidenced by decreased root growth, enhanced ROS production, more membrane damage and Na+ accumulation in sensitive cultivar compared to the tolerant cultivar. Conversely, combined treatments of 2,4-D and saline stress significantly alleviated the growth inhibition and oxidative stress in roots of rice cultivars by modulating lignin and callose deposition, redox states of AsA, GSH, and related enzyme activities involved in the antioxidant defense system. The expression analysis of nine cation transporter genes showed altered and differential gene expression in salt-stressed roots of sensitive and resistant cultivars. Together, these results suggest that 2,4-D differentially regulates the Na+ and K+ levels, ROS production, antioxidant defense, anatomical changes and cation transporters/genes in roots of rice cultivars.

Reduced Glutathione Mediates Pheno-Ultrastructure, Kinome and Transportome in Chromium-Induced Brassica napus L.

Chromium (Cr) as a toxic metal is widely used for commercial purposes and its residues have become a potential environmental threat to both human and plant health. Oilseed rape (Brassica napus L.) is one of the candidate plants that can absorb the considerable quantity of toxic metals from the soil. Here, we used two cultivars of B. napus cvs. ZS 758 (metal-tolerant) and Zheda 622 (metal-susceptible) to investigate the phenological attributes, cell ultrastructure, protein kinases (PKs) and molecular transporters (MTs) under the combined treatments of Cr stress and reduced glutathione (GSH). Seeds of these cultivars were grown in vitro at different treatments i.e., 0, 400 µM Cr, and 400 µM Cr + 1 mM GSH in control growth chamber for 6 days. Results had confirmed that Cr significantly reduced the plant length, stem and root, and fresh biomass such as leaf, stem and root. Cr noticeably caused the damages in leaf mesophyll cells. Exogenous application of GSH significantly recovered both phenological and cell structural damages in two cultivars under Cr stress. For the PKs, transcriptomic data advocated that Cr stress alone significantly increased the gene expressions of BnaA08g16610D, BnaCnng19320D, and BnaA08g00390D over that seen in controls (Ck). These genes encoded both nucleic acid and transition metal ion binding proteins, and protein kinase activity (PKA) and phosphotransferase activities in both cultivars. Similarly, the presence of Cr revealed elite MT genes [BnaA04g26560D, BnaA02g28130D, and BnaA02g01980D (novel)] that were responsible for water transmembrane transporter activity. However, GSH in combination with Cr stress significantly up-regulated the genes for PKs [such as BnaCnng69940D (novel) and BnaC08g49360D] that were related to PKA, signal transduction, and oxidoreductase activities. For MTs, BnaC01g29930D and BnaA07g14320D were responsible for secondary active transmembrane transporter and protein transporter activities that were expressed more in GSH treatment than either Ck or Cr-treated cells. In general, it can be concluded that cultivar ZS 758 is more tolerant toward Cr-induced stress than Zheda 622.

Comparative transcriptome profiling of two Brassica napus cultivars under chromium toxicity and its alleviation by reduced glutathione.

BACKGROUND: Chromium (Cr) being multifarious industrial used element, is considered a potential environmental threat. Cr found to be a prospective water and soil pollutant, and thus it is a current area of concern. Oilseed rape (Brassica napus L.) is well known as a major source of edible oil around the globe. Due to its higher growth, larger biomass and capability to uptake toxic materials B. napus is considered a potential candidate plant against unfavorable conditions. To date, no study has been done that described the Cr and GSH mechanism at RNA-Seq level. RESULTS: Both digital gene expression (DGE) and transcriptome profile analysis (TPA) approaches had opened new insights to uncover the several number of genes related to Cr stress and GSH alleviating mechanism in two leading cultivars (ZS 758 and Zheda 622) of B. napus plants. Data showed that Cr inhibited KEGG pathways i.e. stilbenoid, diarlyheptanoid and gingerol biosynthesis; limonene and pentose degradation and glutathione metabolism in ZS 758; and ribosome and glucosinolate biosynthesis in Zheda-622. On the other hand, vitamin B6, tryptophan, sulfur, nitrogen and fructose and manose metabolisms were induced in ZS 758, and zeatin biosynthesis, linoleic acid metabolism, arginine and proline metabolism, and alanine, asparate and glutamate metabolism pathways in Zheda 622. Cr increased the TFs that were related to hydralase activity, antioxidant activity, catalytic activity phosphatase and pyrophosphatase activity in ZS 758, and vitamin binding and oxidoreductase activity in Zheda 622. Cr also up-regulated the promising proteins related to intracellular membrane bounded organelles, nitrile hyrdatase activity, cytoskeleton protein binding and stress response. It also uncovered, a novel Cr-responsive protein (CL2535.Contig1_All) that was statistically increased as compared to control and GSH treated plants. Exogenously applied GSH successfully not only recovered the changes in metabolic pathways but also induced cysteine and methionine metabolism in ZS 758 and ubiquinone and other terpenoid-quinone biosynthesis pathways in Zheda 622. Furthermore, GSH increased the level of TFs i.e. the gene expression of antioxidant and catalytic activities, iron ion binding and hydrolase activity as compared with Cr. Moreover, results pointed out a novel GSH responsive protein (CL827.Contig3_All) whose expression was found to be significantly increased when compared than Cr stress. Results further delineated that GSH induced TFs such as glutathione disulphide oxidoreducatse and aminoacyl-tRNA ligase activity, and beta glucosidase activity in ZS 758. Similarly in Zheda 622, GSH induced the TFs for instance DNA binding and protein dimerization activity. GSH also highlighted the proteins that were involved in transportation, photosynthesis process, RNA polymerase activity, and against the metal toxicity. These results indicated that cultivar ZS 758 had better metabolism and showed higher tolerance against Cr toxicity. CONCLUSION: The responses of ZS 758 and Zheda 622 differed considerably at both physiological and transcriptional level. Moreover, RNA-Seq method explored the hazardous behavior of Cr as well as GSH up-regulating mechanism by activating plant metabolism, stress responsive genes, TFs and protein encyclopedia.

Differential subcellular distribution and chemical forms of cadmium and copper in Brassica napus.

Metal subcellular fractions and chemical profile highly reflect their level of toxicity to plants. Cadmium and Cu, two different but potentially toxic metals, were compared in the present study for their subcellular distribution and chemical forms in two Brassica napus cultivars (Zheda 622 and ZS 758). Five-week-old seedlings were hydroponically exposed to metal stress and analyzed after 15 days of treatment. In both cultivars, Cd was less retained at cell wall, thus major part of Cd accumulated in the soluble fraction. By contrast, handsome amount of Cu was sequestrated in both cell wall and vacuole containing fraction. Across sensitive organelles, Cu preferentially accumulated in chloroplasts, while Cd was equally distributed in chloroplasts and mitochondria; the two metals intruded nucleus at lesser degree. Further, Cd and Cu differentially interacted with various cellular ligands, and the extent of interaction was higher in the tolerant cultivar ZS 758. Copper was remarkably sequestrated by phosphates, and secondarily by peptide-ligands; inversely, the role of phosphates was secondary in Cd complexation, which was mainly achieved by peptide-ligands. Additional amount of Cu was aggregated with oxalates, but oxalate-bound Cd was scarcely detected. Current results have demonstrated varied toxicological and detoxification pathways of Cd and Cu in B. napus, suggesting that the efficiency of different alleviation strategies could vary against Cd and Cu toxicity to plants.

Salicylic acid mediates antioxidant defense system and ABA pathway related gene expression in Oryza sativa against quinclorac toxicity.

The auxin herbicide quinclorac is widely used for controlling weeds in transplanted and direct-seeded rice fields. However, its phytotoxic responses on rice are still unknown. Therefore, in the present investigation we studied the effects of different concentrations (0, 0.1 and 0.5g/L) of quinclorac herbicide on the physiological and biochemical changes of two rice cultivars (XS 134 and ZJ 88) and further analyzed the ameliorating role of salicylic acid (SA) on quinclorac toxicity in rice plants. The results revealed that exogenous application of SA significantly increased plant biomass and total chlorophyll contents in herbicide stressed plants. The lipid peroxidation and ROS (H2O2, O2(-.), (-)OH) production were significantly increased in roots and leaves of both rice cultivars under quinclorac stress, demonstrating an oxidative burst in rice plants. Whereas, application of SA significantly lowered ROS contents under quinclorac stress. Further, exogenous SA treatment significantly modulated antioxidant enzymes and enhanced GSH concentration in stress plants. Anatomical observations of leaf and root revealed that herbicide affected internal structures, while SA played a vital role in protection from toxic effects. Expression analysis of stress hormone ABA genes (OsABA8oxs, OsNCEDs) revealed that quinclorac application enhanced stress condition in cultivar ZJ 88, while SA treatment downregulated ABA genes more in cultivar XS 134, which correlated with the enhanced tolerance to quinclorac induced oxidative stress in this cultivar. The present study delineated that SA played a critical role under quinclorac stress in both rice cultivars by regulating antioxidant defense system, reducing ROS formation and preventing the degradation of internal cell organelles.

Role of exogenous salicylic acid in regulating physio-morphic and molecular changes under chromium toxicity in black- and yellow- seeded Brassica napus L.

Salicylic acid (SA) mediates tolerance mechanisms in plants against a wide spectrum of biotic and abiotic stresses. Therefore, the present study was carried out to determine how SA regulates the plant protection mechanisms in two cultivars of oilseed rape (Brassica napus L.) under chromium (Cr) stress. Exogenously applied SA enhanced plant growth, increased dry biomasses, and strengthened the reactive oxygen scavenging system by improving cell organelles that were severely damaged via Cr toxicity. The contents of Cr were significantly enhanced in both root and leaf of cultivar Zheda 622 (yellow color) compared with cultivar ZS 758 (black color). Exogenous application of SA significantly reduced the Cr contents in both plant organs as well as enhanced the SA contents under Cr stress. A dose-dependent increase was observed in reactive oxygen species (ROS) generation under Cr stress. To ease the inimical effects of ROS, plants' defense systems were induced under Cr stress, and SA further enhanced protection. Further, TEM micrographs results showed that Cr stress alone significantly ruptured the plant cell organelles of both cultivars by increasing the size of starch grain and the number of plastoglobuli, damaging the chloroplast and mitochondrion structures. However, exogenously applied SA significantly recovered these damages in the plant cells of both cultivars. It was also observed that cultivar ZS 758 was proved to be more tolerant under Cr toxicity. Gene expression analysis revealed that combined treatments of Cr and SA increased antioxidant-related gene expression in both cultivars. Findings of the present study demonstrate that SA induces the enzymatic antioxidant activities and related gene expression, secondary metabolism, and improves the cell structural changes and the transcript level of specific stress-associated proteins in root and leaf of two oilseed rape cultivars under Cr toxicity.

Combined herbicide and saline stress differentially modulates hormonal regulation and antioxidant defense system in Oryza sativa cultivars.

Plants are simultaneously exposed to a combination of biotic and abiotic stresses in field conditions. Crops respond to the combined stress in a unique way which cannot be understood by extrapolating the results of individual stress. In the present study, effects of individual and combined stress of herbicide (2,4-dichlorophenoxyacetic acid) and salinity (NaCl) on two Oryza sativa cultivars (ZJ 88 and XS 134) were investigated. Both herbicide and saline stress affected the plant growth differentially and produced oxidative stress in rice cultivars. Interestingly, the combination of herbicide and salinity showed a significant protection to both rice cultivars by reducing ROS (H2O2, O2(-)) and lipid peroxidation through modulation of enzymatic (SOD, POD, CAT and APX) and non-enzymatic (TSP, sugars, phenolic and proline) antioxidants. In addition, active regulation of transcript levels of genes encoding Na(+) and K(+) (OsHKT1;5, OsLti6a,b, OsHKT2;1, OsSOS1, OsCNGC1, OsNHX1 and OsAKT1) transporter proteins reduced sodium and enhanced potassium accumulation under combined stress, resulted a better growth and ionic homeostasis in both rice cultivars. The production of ABA and IAA was significantly higher in cultivar XS 134 compared to cultivar ZJ 88 under control conditions. However, combined herbicide and saline stress enhanced the accumulation of phytohormones (IAA and ABA) and transcription of ethylene in cultivar ZJ 88, which might be one of the factors responsible for poor salt tolerance in sensitive cultivar. These findings indicated that herbicide application under saline stress confers tolerance to salinity in rice cultivars, likely by reducing oxidative damage, modulating mineral absorption, upgradation of antioxidant defense and by dynamic regulation of key genes involved in Na(+) and K(+) homeostasis in plants.

Methyl Jasmonate Regulates Antioxidant Defense and Suppresses Arsenic Uptake in Brassica napus L.

Methyl jasmonate (MJ) is an important plant growth regulator, involved in plant defense against abiotic stresses, however, its possible function in response to metal stress is poorly understood. In the present study, the effect of MJ on physiological and biochemical changes of the plants exposed to arsenic (As) stress were investigated in two Brassica napus L. cultivars (ZS 758 - a black seed type, and Zheda 622 - a yellow seed type). The As treatment at 200 µM was more phytotoxic, however, its combined application with MJ resulted in significant increase in leaf chlorophyll fluorescence, biomass production and reduced malondialdehyde content compared with As stressed plants. The application of MJ minimized the oxidative stress, as revealed via a lower level of reactive oxygen species (ROS) synthesis (H2O2 and OH(-)) in leaves and the maintenance of high redox states of glutathione and ascorbate. Enhanced enzymatic activities and gene expression of important antioxidants (SOD, APX, CAT, POD), secondary metabolites (PAL, PPO, CAD) and induction of lypoxygenase gene suggest that MJ plays an effective role in the regulation of multiple transcriptional pathways which were involved in oxidative stress responses. The content of As was higher in yellow seeded plants (cv. Zheda 622) as compared to black seeded plants (ZS 758). The application of MJ significantly reduced the As content in leaves and roots of both cultivars. Findings of the present study reveal that MJ improves ROS scavenging through enhanced antioxidant defense system, secondary metabolite and reduced As contents in both the cultivars.

Subcellular distribution, modulation of antioxidant and stress-related genes response to arsenic in Brassica napus L.

Arsenic (As) is an environmental toxin pollutant that affects the numerous physiological processes of plants. In present study, two Brassica napus L. cultivars were subjected to various concentrations (0, 50, 100, and 200 µM) of As for 14 days, plants were examined for As subcellular distribution, photosynthesis parameters, oxidative stress, and ultrastructural changes under As-stress. Differential fraction analysis showed that significant amount of As was accumulated in the cell wall as compared to other organelles. Decline in photosynthetic efficiency under As stress was observed in term of reduced pigment contents and gas exchange parameters. Differential responses of antioxidants at both enzymatic and gene levels to higher As stress were more pronounced in cultivar ZS 758 as compared to Zheda 622. The qRT-PCR analysis showed that heat shock protein 90 (Hsp90) and metallothionein were over-expressed in As stressed B. napus plants. Disorganization of cell structure and the damages in different organelles were some of the obvious variations in cultivar Zheda 622 as compared to ZS 758.

Physiological and molecular analyses of black and yellow seeded Brassica napus regulated by 5-aminolivulinic acid under chromium stress.

Brassica napus L. is a promising oilseed crop among the oil producing species. So, it is prime concern to screen the metal tolerant genotypes in order to increase the oilseed rape production through the utilization of pollutant soil regimes. Nowadays, use of plant growth regulators against abiotic stress is one of the major objectives of researchers. In this study, an attempt was carried out to analyze the pivotal role of exogenously applied 5-amenolevulinic acid (ALA) on alleviating chromium (Cr)-toxicity in black and yellow seeded B. napus. Plants of two cultivars (ZS 758 - a black seed type, and Zheda 622 - a yellow seed type) were treated with 400 µM Cr with or without 15 and 30 mg/L ALA. Results showed that exogenously applied ALA improved the plant growth and increased ALA contents; however, it decreased the Cr concentration in B. napus leaves under Cr-toxicity. Moreover, exogenous ALA reduced oxidative stress by up-regulating antioxidant enzyme activities and their related gene expression. Further, results suggested that stress responsive protein's transcript level such as HSP90-1 and MT-1 were increased under Cr stress alone in both cultivars. Exogenously applied ALA further enhanced the expression rate in both genotypes and obviously results were found in favor of cultivar ZS 758. The ultrastructural changes were observed more obvious in yellow seeded than black seeded cultivar; however, exogenously applied ALA helped the plants to recover their cell turgidity under Cr stress. The present study describes a detailed molecular mechanism how ALA regulates the plant growth by improving antioxidant machinery and related transcript levels, cellular modification as well as stress related genes expression under Cr-toxicity.

Regulation of Cadmium-Induced Proteomic and Metabolic Changes by 5-Aminolevulinic Acid in Leaves of Brassica napus L.

It is evident from previous reports that 5-aminolevulinic acid (ALA), like other known plant growth regulators, is effective in countering the injurious effects of heavy metal-stress in oilseed rape (Brassica napus L.). The present study was carried out to explore the capability of ALA to improve cadmium (Cd2+) tolerance in B. napus through physiological, molecular, and proteomic analytical approaches. Results showed that application of ALA helped the plants to adjust Cd2+-induced metabolic and photosynthetic fluorescence changes in the leaves of B. napus under Cd2+ stress. The data revealed that ALA treatment enhanced the gene expressions of antioxidant enzyme activities substantially and could increase the expression to a certain degree under Cd2+ stress conditions. In the present study, 34 protein spots were identified that differentially regulated due to Cd2+ and/or ALA treatments. Among them, 18 proteins were significantly regulated by ALA, including the proteins associated with stress related, carbohydrate metabolism, catalysis, dehydration of damaged protein, CO2 assimilation/photosynthesis and protein synthesis/regulation. From these 18 ALA-regulated proteins, 12 proteins were significantly down-regulated and 6 proteins were up-regulated. Interestingly, it was observed that ALA-induced the up-regulation of dihydrolipoyl dehydrogenase, light harvesting complex photo-system II subunit 6 and 30S ribosomal proteins in the presence of Cd2+ stress. In addition, it was also observed that ALA-induced the down-regulation in thioredoxin-like protein, 2, 3-bisphosphoglycerate, proteasome and thiamine thiazole synthase proteins under Cd2+ stress. Taken together, the present study sheds light on molecular mechanisms involved in ALA-induced Cd2+ tolerance in B. napus leaves and suggests a more active involvement of ALA in plant physiological processes than previously proposed.

Oxidative injury and antioxidant enzymes regulation in arsenic-exposed seedlings of four Brassica napus L. cultivars.

Environmental contamination due to arsenic (As) has become a major risk throughout the world; this affects plant growth and productivity. Its accumulation in food chain may pose a severe threat to organisms. The present study was carried out to observe the toxic effects of As (0, 50, 100, and 200 µM) on physiological and biochemical changes in four Brassica napus cultivars (ZS 758, Zheda 619, ZY 50, and Zheda 622). Results showed that As toxicity provoked a significant inhibition in growth parameters of B. napus cultivars and this reduction was more obvious in cultivar Zheda 622. The highest concentration of MDA, H2O2, and O2 (-) contents in both leaf and root tissues were observed at 200 µM As level, and a gradual decrease was observed at lower concentrations. Increasing As concentration gradually decreased chlorophyll and carotenoids contents. Activity of antioxidant enzymes such as SOD, CAT, APX, GR, and GSH was positively correlated with As treatments in all cultivars. The microscopic study of leaves and roots at 200 µM As level showed the disorganization in cell organelles. Disturbance in the morphology of chloroplast, broken cell wall, increase in size, and number of starch grains and immature nucleus were found in leaf ultrastructures under higher concentration of As. Moreover, damaged nucleus, diffused cell wall, enlarged vacuoles, and a number of mitochondria were observed in root tip cells at 200 µM As level. These results suggest that B. napus cultivars have efficient mechanism to tolerate As toxicity, as evidenced by an increased level of antioxidant enzymes.