Three species of rape responded to cadmium and melatonin alleviating Cd-toxicity in species-specific strategy.

Cadmium (Cd) pollution has been a significant concern in heavy metal pollution, prompting plants to adopt various strategies to mitigate its damage. While the response of plants to Cd stress and the impact of exogenous melatonin has received considerable attention, there has been limited focus on the responses of closely related species to these factors. Consequently, our investigation aimed to explore the response of three different species of rape to Cd stress and examine the influence of exogenous melatonin in this scenario. The research findings revealed distinctive responses among the investigated rape species. B. campestris showed the resistance to Cd and exhibited lower Cd absorption and sustained its physiological activity under Cd stress. In contrast, B. juncea accumulated much Cd and increased the amount of anthocyanin to mitigate the Cd-damage. Furthermore, B. napus showed the tolerance to Cd and tended to accumulate Cd in vacuoles under Cd stress, thereby decreasing the Cd damage and leading to higher activity of antioxidant enzymes and photosynthesis. Moreover, the application of exogenous melatonin significantly elevated the melatonin level in plants and mitigated Cd toxicity by promoting the activity of antioxidant enzymes, reducing Cd absorption, enhancing the chelating capacity with Cd, decreasing Cd accumulation in organelles, and reducing its fluidity. Specifically, exogenous melatonin increased the FHAc content in B. campestris, elevated the phytochelatins (PCs) level in B. napus, and stimulated photosynthesis in B. juncea. In summary, the findings underscore the species-specific responses of the three species of rape to both Cd stress and exogenous melatonin, highlighting the potential for tailored mitigation strategies based on the unique characteristics of each species.

Deciphering proteomic mechanisms explaining the role of glutathione as an aid in improving plant fitness and tolerance against cadmium-toxicity in Brassica napus L.

Cadmium (Cd) hazard is a serious limitation to plants, soils and environments. Cd-toxicity causes stunted growth, chlorosis, necrosis, and plant yield loss. Thus, ecofriendly strategies with understanding of molecular mechanisms of Cd-tolerance in plants is highly demandable. The Cd-toxicity caused plant growth retardation, leaf chlorosis and cellular damages, where the glutathione (GSH) enhanced plant fitness and Cd-toxicity in Brassica through Cd accumulation and antioxidant defense. A high-throughput proteome approach screened 4947 proteins, wherein 370 were differently abundant, 164 were upregulated and 206 were downregulated. These proteins involved in energy and carbohydrate metabolism, CO2 assimilation and photosynthesis, signal transduction and protein metabolism, antioxidant defense response, heavy metal detoxification, cytoskeleton and cell wall structure, and plant development in Brassica. Interestingly, several key proteins including glutathione S-transferase F9 (A0A078GBY1), ATP sulfurylase 2 (A0A078GW82), cystine lyase CORI3 (A0A078FC13), ferredoxin-dependent glutamate synthase 1 (A0A078HXC0), glutaredoxin-C5 (A0A078ILU9), glutaredoxin-C2 (A0A078HHH4) actively involved in antioxidant defense and sulfur assimilation-mediated Cd detoxification process confirmed by their interactome analyses. These candidate proteins shared common gene networks associated with plant fitness, Cd-detoxification and tolerance in Brassica. The proteome insights may encourage breeders for enhancing multi-omics assisted Cd-tolerance in Brassica, and GSH-mediated hazard free oil seed crop production for global food security.

Combined application of zinc and iron-lysine and its effects on morpho-physiological traits, antioxidant capacity and chromium uptake in rapeseed (Brassica napus L.).

Environmental contamination of chromium (Cr) has gained substantial consideration worldwide because of its high levels in the water and soil. A pot experiment using oil seed crop (rapeseed (Brassica napus L.)) grown under different levels of tannery wastewater (0, 33, 66 and 100%) in the soil using the foliar application of zinc (Zn) and iron (Fe)-lysine (lys) has been conducted. Results revealed that a considerable decline in the plant growth and biomass elevates with the addition of concentrations of tannery wastewater. Maximum decline in plant height, number of leaves, root length, fresh and dry biomass of root and leaves were recorded at the maximum level of tannery wastewater application (100%) compared to the plants grown without the addition of tannery wastewater (0%) in the soil. Similarly, contents of carotenoid and chlorophyll, gas exchange parameters and activities of various antioxidants (superoxidase dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX)) were also reduced significantly (P < 0.05) with the increasing concentration of tannery wastewater (33, 66 and 100%) in the soil. In addition, a combined application of Zn and Fe-lys reduced the accumulation and uptake of toxic Cr, while boosting the uptake of essential micronutrients such as Zn and Fe in different tissues of the plants. Results concluded that exogenous application of micronutrients chelated with amino acid successfully mitigate Cr stress in B. napus. Under field conditions, supplementation with these micronutrient-chelated amino acids may be an effective method for alleviating metal stress in other essential seed crops.

Potential roles for pattern molecule of PAMP-triggered immunity in improving crop cold tolerance.

KEY MESSAGE: The application of flagellin 22 (flg22), the most widely studied PAMP, enhance crop cold tolerance. ICE1-CBF pathway and SA signaling is involved in the alleviation of cold injury by flg22 treatment. Pathogen infection cross-activates cold response and increase cold tolerance of host plants. However, it is not possible to use the infection to increase cold tolerance of field plants. Here flagellin 22 (flg22), the most widely studied PAMP (pathogen-associated molecular patterns), was used to mimic the pathogen infection to cross-activate cold response. Flg22 treatment alleviated the injury caused by freezing in Arabidopsis, oilseed and tobacco. In Arabidopsis, flg22 activated the expression of immunity and cold-related genes. Moreover, the flg22 induced alleviation of cold injury was lost in NahG transgenic line (SA-deficient), sid2-2 and npr1-1 mutant plants, and flg22-induced expression of cold tolerance-related genes, which indicating that salicylic acid signaling pathway is required for the alleviation of cold injury by flg22 treatment. In short flg22 application can be used to enhance cold tolerance in field via a salicylic acid-depended pathway.

Role of beneficial microbes with nitrogen and phosphorous levels on canola productivity / Papel de microbes benéficos em associação com níveis de nitrogênio e fósforo na produtividade de canola

A research was conducted to evaluate the impact of various nitrogen and phosphorus levels along with beneficial microbes to enhance canola productivity. The research was carried out at Agronomy Research Farm, The University of Agriculture Peshawar in winter 2016-2017. The experiment was conducted in randomized complete block factorial design. The study was comprised of three factors including nitrogen (60, 120 and 180 kg ha-¹), phosphorous (70, 100 and 130 kg ha-¹) and beneficial microbes (with and without BM). A control treatment with no N, P and BM was also kept for comparison. Application of beneficial microbes significantly increased pods plant, seed pod, seed filling duration, 1000 seed weight, biological yield and seed yield as compared to control plots. Nitrogen applied at the rate of 180 kg ha-¹ increased pods plant-¹, seed pod, seed filling duration, seed weight, biological yield and seed yield. Maximum pods plant-¹, seed pod, early seed filling, heavier seed weight, biological yield, seed yield, and harvest index were observed in plots treated with 130 kg.ha-¹ phosphorous. As comparison, the combine treated plots have more pods plant-¹, seeds pod-¹, seed filling duration, heaviest seeds, biological yield, seed yield and harvest index as compared to control plots. It is concluded that application of beneficial microbes with N and P at the rate of 180 kg ha-¹ and 130 kg ha-¹, respectively, increased yield and its attributes for canola.

Uma pesquisa foi realizada para avaliar o impacto de vários níveis de nitrogênio e fósforo, juntamente com micróbios benéficos, para aumentar a produtividade da canola. A pesquisa foi realizada no inverno de 2016-17 no Agronomy Research Farm, Universidade de Agricultura do Peshawar. O experimento foi conduzido por planejamento fatorial aleatorizado em blocos. O estudo focou-se em três fatores, incluindo o teor de nitrogênio, N, (60, 120 e 180 kg.ha-¹), o teor de fósforo, P, (70, 100 e 130 kg ha-¹) e a presença de micróbios benéficos (com BM e sem BM). Para fins de comparação, um tratamento controle sem N, P e BM também foi incluído no estudo. A aplicação de micróbios benéficos aumentou significativamente as vagens das plantas e de sementes, a duração do enchimento das sementes, o peso de 1000 sementes, o rendimento biológico e o rendimento de sementes em comparação com os resultados do controle. O nitrogênio aplicado na taxa de 180 kg ha-¹ aumentou as vagens por planta, vagem, duração do enchimento, peso da semente, rendimento biológico e rendimento de sementes. Vagens máximas por planta, vagem, enchimento precoce de sementes, peso maior de semente, rendimento biológico, rendimento de sementes e índice de colheita foram observados em parcelas tratadas com 130 kg.ha-¹ de fósforo. Em comparação aos blocos cultivados de controle, os blocos cultivados tratados combinados têm mais vagens por planta e sementes por vagem, maior duração do enchimento das sementes, maior número de sementes mais pesadas e maior rendimento biológico, rendimento de sementes e índice de colheita. Conclui-se que a aplicação de micróbios benéficos junto com N e P nas doses de 180 kg ha-¹ e 130 kg ha-¹, respectivamente, aumentou a produtividade e atributos de produtividade para a canola.

Exploration of physiological and biochemical processes of canola with exogenously applied fertilizers and plant growth regulators under drought stress.

Environmental stresses may alter the nutritional profile and economic value of crops. Chemical fertilizers and phytohormones are major sources which can enhance the canola production under stressful conditions. Physio-biochemical responses of canola altered remarkably with the use of nitrogen/phosphorus/potassium (N/P/K) fertilizers and plant growth regulators (PGRs) under drought stress. The major aim of current study was to evaluate nutritional quality and physio-biochemical modulation in canola (Brassica napus L.) from early growth to seed stage with NPK and PGRs in different water regimes. To monitor biochemical and physiological processes in canola, two season field experiment was conducted as spilt plot under randomized complete block design (RCBD) with four treatments (Control, Chemical fertilizers [N (90 kg/ha), P and K (45 kg ha-1)], PGRs; indole acetic acid (IAA) 15g ha-1, gibberellic acid (GA3) 15g ha-1 and the combination of NPK and PGRs] under different irrigations regimes (60, 100, 120, 150 mm evaporations). Water stress enhanced peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), polyphenol oxidase (PPO), soluble sugar, malondialdehyde (MDA), proline contents as well as leaf temperature while substantially reduced leaf water contents (21%), stomatal conductance (50%), chlorophyll contents (10-67%), membrane stability index (24%) and grain yield (30%) of canola. However, the combined application of NPK and PGR further increased the enzymatic antioxidant pool, soluble sugars, along with recovery of leaf water contents, chlorophyll contents, stomatal conductance and membrane stability index but decreased the proline contents and leaf temperature at different rate of evaporation. There is positive interaction of applied elicitors to the water stress in canola except leaf area. The outcomes depicted that the combination of NPK with PGRs improved the various morpho-physiological as well as biochemical parameters and reduced the pressure of chemical fertilizers cost about 60%. It had also reduced the deleterious effect of water limitation on the physiology and grain yield and oil contents of canola in field experiments.

Effects of nickel on morpho-physiological parameters and oxidative status in Brassica napus cultivars under different sulphur levels.

Increased levels of trace metals are an important problem of environmental pollution. Ni is one of the metals essential for normal plant development, but elevated levels usually cause deleterious effects on plant growth. The aim of the study was to evaluate the effects sulphur nutrition on growth, oxidative status, and Ni bioaccumulation of Ni-treated rape (Brassica napus L.). Two different oilseed rape cultivars (Hammer and Compass) were grown under sulphur deficiency and under optimal S availability (0 and 1 mM sulphate, respectively) and exposed to 0.1, 0.3, and 0.5 mM Ni concentrations for 3 weeks. Exposure of plants to elevated Ni concentrations resulted in a decrease in the shoot and root biomass and chlorophyll content. The enhancement of Ni caused increased lipid peroxidation. The sulphur nutrition had an effect on the level of oxidative stress of Ni-treated plants-under the deficiency of sulphur the concentration of TBARS was significantly higher than under the optimal level of S. The beneficial effect of optimal sulphur nutrition was lower Ni accumulation in exposed plants but translocation of Ni was dependent on the cultivar.

Ectopic overexpression of a membrane-tethered transcription factor gene NAC60 from oilseed rape positively modulates programmed cell death and age-triggered leaf senescence.

Senescence is an integrative final stage of plant development that is governed by internal and external cues. The NAM, ATAF1/2, CUC2 (NAC) transcription factor (TF) family is specific to plants and membrane-tethered NAC TFs (MTTFs) constitute a unique and sophisticated mechanism in stress responses and development. However, the function of MTTFs in oilseed rape (Brassica napus L.) remains unknown. Here, we report that BnaNAC60 is an MTTF associated with the endoplasmic reticulum (ER) membrane. Expression of BnaNAC60 was induced during the progression of leaf senescence. Translocation of BnaNAC60 into nuclei was induced by ER stress and oxidative stress treatments. It binds to the NTLBS motif, rather than the canonical NAC recognition site. Overexpression of BnaNAC60 devoid of the transmembrane domain, but not the full-length BnaNAC60, induces significant reactive oxygen species (ROS) accumulation and hypersensitive response-like cell death in both tobacco (Nicotiana benthamiana) and oilseed rape protoplasts. Moreover, ectopic overexpression of BnaNAC60 devoid of the transmembrane domain, but not the full-length BnaNAC60, in Arabidopsis also induces precocious leaf senescence. Furthermore, screening and expression profiling identified an array of functional genes that are significantly induced by BnaNAC60 expression. Further it was found that BnaNAC60 can activate the promoter activities of BnaNYC1, BnaRbohD, BnaBFN1, BnaZAT12, and multiple BnaVPEs in a dual-luciferase reporter assay. Electrophoretic mobility shift assay and chromatin immunoprecipitation coupled to quantitative PCR assays revealed that BnaNAC60 directly binds to the promoter regions of these downstream target genes. To summarize, our data show that BnaNAC60 is an MTTF that modulates cell death, ROS accumulation, and leaf senescence.

Nano zinc elicited biochemical characterization, nutritional assessment, antioxidant enzymes and fatty acid profiling of rapeseed.

The use of nanomaterials in agriculture is a current need and could be helpful in overcoming food security risks. Brassica napus L. is the third most important crop for edible oil, having double low unsaturated fatty acids. In the present study, we investigated the effects of green synthesized Zn NPs on biochemical effects, antioxidant enzymes, nutritional quality parameters and on the fatty acid profile of rapeseed (B. napus). Plant-mediated synthesis of zinc nanoparticles (Zn NPs) was carried out using Mentha arvensis L. leaf extract followed by characterization through ultraviolet-visible spectroscopy (UV-vis), scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive X-Ray (EDX), and X-Ray diffraction (XRD). NPs exhibited irregular shapes ranging in size from 30-70 nm and EDX analysis confirmed 96.08% of Zn in the sample. The investigated biochemical characterization (protein content, proline content, total soluble sugar (TSS), total flavonoid content (TFC), and total phenolic content (TPC) showed a substantial change on exposure to Zn NPs. A dose-dependent gradual increase was observed in the antioxidant enzymes, superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Oil and moisture contents dropped significantly from the control level in the rapeseed (B. napus) varieties. However, different trends in nutritional (Zn, Na+, K+) and fatty acid profiling of B. napus have been noted. This study demonstrates that Zn NPs have the potential to improve the biochemical, nutritional, antioxidant enzymes, and fatty acid profile of B. napus varieties.

Development and screening of EMS mutants with altered seed oil content or fatty acid composition in Brassica napus.

Brassica napus is an important oilseed crop in the world, and the mechanism of seed oil biosynthesis in B. napus remains unclear. In order to study the mechanism of oil biosynthesis and generate germplasms for breeding, an ethyl methanesulfonate (EMS) mutant population with ~100 000 M2 lines was generated using Zhongshuang 11 as the parent line. The EMS-induced genome-wide mutations in M2-M4 plants were assessed. The average number of mutations including single nucleotide polymorphisms and insertion/deletion in M2-M4 was 21 177, 28 675 and 17 915, respectively. The effects of the mutations on gene function were predicted in M2-M4 mutants, respectively. We screened the seeds from 98 113 M2 lines, and 9415 seed oil content and fatty acid mutants were identified. We further confirmed 686 mutants with altered seed oil content and fatty acid in advanced generation (M4 seeds). Five representative M4 mutants with increased oleic acid were re-sequenced, and the potential causal variations in FAD2 and ROD1 genes were identified. This study generated and screened a large scale of B. napus EMS mutant population, and the identified mutants could provide useful genetic resources for the study of oil biosynthesis and genetic improvement of seed oil content and fatty acid composition of B. napus in the future.

Role of iron-lysine on morpho-physiological traits and combating chromium toxicity in rapeseed (Brassica napus L.) plants irrigated with different levels of tannery wastewater.

Chromium (Cr) is among the most widespread toxic trace elements found in agricultural soils resulting from various anthropogenic activities. However, the role of micronutrient-amino acid chelates in reducing Cr toxicity in crop plants has recently been suggested. The present study was conducted to explore the effect of iron (Fe) chelated with lysine (lys) on plant growth, biomass, gaseous exchange attributes, oxidative stress indicators, antioxidant response, and Cr uptake in rapeseed (Brassica napus L.) plants irrigated with different levels of tannery wastewater in soil collected from District Kasur of Pakistan. B. napus seedlings (thirty-day-old) were shifted to pots irrigated with different levels of tannery wastewater. After two weeks, foliar application of Fe-lys (5 mM) was carried out for four successive weeks, and plants were harvested carefully post ten weeks of cultivation in tannery wastewater, under controlled conditions. Toxic levels of Cr in the soil significantly decreased plant height, fresh biomass of roots and leaves, dry biomass of roots and leaves, root length, number of leaves, leaf area, total chlorophyll contents, carotenoid contents, transpiration rate (E), stomatal conductance (gs), net photosynthesis (PN), and water use efficiency (WUE). Toxic Cr levels in the soil also increased oxidative stress in the roots and leaves of B. napus plants, which were overcome by the activities of various antioxidant enzymes, such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX). Moreover, increasing levels of Cr in the soil caused a significant increase in the Cr content of the roots and shoots of B. napus plants. The negative effects of Cr toxicity could be overturned by Fe-lys application, significantly increasing plant growth, biomass, chlorophyll content, and gaseous exchange attributes by reducing oxidative stress (H2O2, MDA, EL) and enhancing antioxidant enzyme activities. Furthermore, foliar application of Fe-lys reduced the Cr concentration and increased essential micronutrients (Fe contents) in the roots and shoots of B. napus plants. These results shed light on the effectiveness of Fe-lys in improving the growth and up-regulation of antioxidant enzyme activities of B. napus in response to Cr stress. However, further studies at field levels are required to explore the mechanisms of Fe-lys-mediated reduction of the toxicity of not only Cr, but possibly also other heavy metals in plants.

Proteasome Inhibition in Brassica napus Roots Increases Amino Acid Synthesis to Offset Reduced Proteolysis.

Cellular homeostasis is maintained by the proteasomal degradation of regulatory and misfolded proteins, which sustains the amino acid pool. Although proteasomes alleviate stress by removing damaged proteins, mounting evidence indicates that severe stress caused by salt, metal(oids), and some pathogens can impair the proteasome. However, the consequences of proteasome inhibition in plants are not well understood and even less is known about how its malfunctioning alters metabolic activities. Lethality causes by proteasome inhibition in non-photosynthetic organisms stem from amino acid depletion, and we hypothesized that plants respond to proteasome inhibition by increasing amino acid biosynthesis. To address these questions, the short-term effects of proteasome inhibition were monitored for 3, 8 and 48 h in the roots of Brassica napus treated with the proteasome inhibitor MG132. Proteasome inhibition did not affect the pool of free amino acids after 48 h, which was attributed to elevated de novo amino acid synthesis; these observations coincided with increased levels of sulfite reductase and nitrate reductase activities at earlier time points. However, elevated amino acid synthesis failed to fully restore protein synthesis. In addition, transcriptome analysis points to perturbed abscisic acid signaling and decreased sugar metabolism after 8 h of proteasome inhibition. Proteasome inhibition increased the levels of alternative oxidase but decreased aconitase activity, most sugars and tricarboxylic acid metabolites in root tissue after 48 h. These metabolic responses occurred before we observed an accumulation of reactive oxygen species. We discuss how the metabolic response to proteasome inhibition and abiotic stress partially overlap in plants.

Physiological and molecular mechanism of cadmium (Cd) tolerance at initial growth stage in rapeseed (Brassica napus L.).

Cadmium (Cd) contaminated soil has threatened plant growth and human health. Rapeseed (Brassica napus L.), an ideal plant for phytoremediation, is an important source of edible vegetable oil, vegetable, animal fodder, green manure and biodiesel. For safe utilization of Cd polluted soil, physiological, biochemical, and molecular techniques have been used to understand mechanisms of Cd tolerance in B. napus. However, most of these researches have concentrated on vegetative and adult stages, just a few reports focus on the initial growth stage. Here, the partitioning of cadmium, gene expression level and activity of enzymatic antioxidants of H18 (tolerant genotype) and P9 (sensitive genotype) were investigated under 0 and 30 mg/L Cd stress at seedling establishment stage. Results shown that the radicle length of H18 and P9 under Cd stress were decreased by 30.33 (0.01 < P < 0.05) and 88.89% (P < 0.01) respectively. Cd concentration at cotyledon not radicle and hypocotyl in P9 was significantly higher than that in H18. The expression level of BnaHMA4c, which plays a key role in root-to-shoot translocation of Cd, was extremely higher in P9 than in H18 under both normal and Cd stress conditions. We also found that SOD, CAT and POD were more active in responding to Cd stress after 48 h, and the activity of SOD and CAT in H18 were higher than that in P9 at all observed time points. In conclusion, high activity of enzymatic antioxidants at initial Cd stress stage is the main detoxification mechanism in Cd-tolerant rapeseed, while the higher Cd transfer coefficient, driven by higher expression level of BnaHMA4c is the main mechanism for surviving radicle from initial Cd toxicity in Cd-sensitive rapeseed.

Melatonin mitigates cadmium and aluminium toxicity through modulation of antioxidant potential in Brassica napus L.

Melatonin has emerged as an essential molecule in plants, due to its role in defence against metal toxicity. Aluminium (Al) and cadmium (Cd) toxicity inhibit rapeseed seedling growth. In this study, we applied different doses of melatonin (50 and 100 µm) to alleviate Al (25 µm) and Cd (25 µm) stress in rapeseed seedlings. Results show that Al and Cd caused toxicity in rapeseed seedling, as evidenced by a decrease in height, biomass and antioxidant enzyme activity. Melatonin increased the expression of melatonin biosynthesis-related Brassica napus genes for caffeic acid O-methyl transferase (BnCOMT) under Al and Cd stress. The genes BnCOMT-1, BnCOMT-5 and BnCOMT-8 showed up-regulated expression, while BnCOMT-4 and BnCOMT-6 were down-regulated during incubation in water. Melatonin application increased the germination rate, shoot length, root length, fresh and dry weight of seedlings. Melatonin supplementation under Al and Cd stress increased superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, proline, chlorophyll and anthocyanin content, as well as photosynthesis rate. Both Cd and Al treatments significantly increased hydrogen peroxide and malondialdehyde levels in rapeseed seedlings, which were strictly counterbalanced by melatonin. Analysis of Cd and Al in different subcellular compartments showed that melatonin enhanced cell wall and soluble fractions, but reduced the vacuolar and organelle fractions in Al- and Cd-treated seedlings. These results suggest that melatonin-induced improvements in antioxidant potential, biomass, photosynthesis rate and successive Cd and Al sequestration play a pivotal role in plant tolerance to Al and Cd stress. This mechanism may have potential implications in safe food production.

MicroRNA-mRNA expression profiles and their potential role in cadmium stress response in Brassica napus.

BACKGROUND: Oilseed rape is an excellent candidate for phytoremediation of cadmium (Cd) contaminated soils given its advantages of high biomass, fast growth, moderate metal accumulation, ease of harvesting, and metal tolerance, but the cadmium response pathways in this species (Brassica napus) have yet to be fully elucidated. A combined analysis of miRNA and mRNA expression to infer Cd-induced regulation has not been reported in B. napus. RESULTS: We characterized concurrent changes in miRNA and mRNA profiles in the roots and shoots of B. napus seedlings after 10 days of 10 mg/L Cd2+ treatment. Cd treatment significantly affected the expression of 22 miRNAs belonging to 11 families in the root and 29 miRNAs belonging to 14 miRNA families in the shoot. Five miRNA families (MIR395, MIR397, MIR398, MIR408 and MIR858) and three novel miRNAs were differentially expressed in both tissues. A total of 399 differentially expressed genes (DEGs) in the root and 389 DEGs in the shoot were identified, with very little overlap between tissue types. Eight anti-regulation miRNA-mRNA interaction pairs in the root and eight in the shoot were identified in response to Cd and were involved in key plant stress response pathways: for example, four genes targeted by miR398 were involved in a pathway for detoxification of superoxide radicals. Cd stress significantly impacted the photosynthetic pathway. Transcription factor activation, antioxidant response pathways and secondary metabolic processes such as glutathione (GSH) and phenylpropanoid metabolism were identified as major components for Cd-induced response in both roots and shoots. CONCLUSIONS: Combined miRNA and mRNA profiling revealed miRNAs, genes and pathways involved in Cd response which are potentially critical for adaptation to Cd stress in B. napus. Close crosstalk between several Cd-induced miRNAs and mRNAs was identified, shedding light on possible mechanisms for response to Cd stress in underground and aboveground tissues in B. napus. The pathways, genes, and miRNAs identified here will be valuable targets for future improvement of cadmium tolerance in B. napus.

Selenium mitigates the chromium toxicity in Brassicca napus L. by ameliorating nutrients uptake, amino acids metabolism and antioxidant defense system.

The phytotoxicity of chromium (Cr) makes it obligatory for the researchers to develop strategies that seek to hinder its accumulation in food chains. While, protective role of selenium (Se) has not been discussed in detail under adverse conditions in oilseed rape. Here, our aim was to investigate the potential use of Se (0, 5 and 10 µM) in alleviating the Cr toxicity (0, 100 and 200 µM) in Brassica napus L. Results delineated that Se-supplementation notably recovered the Cr-phytotoxicity by reducing the Cr accumulation in plant tissues and boosted the inhibition in plant growth and biomass. Under Cr stress, the exogenously applied Se significantly recovered the impairment in photosynthesis related parameters (chlorophyll a, chlorophyll b, carotenoids, net photosynthetic rate, stomatal conductance, and photochemical efficiency of photosystem II), and counteracted the reduction in nutrients uptake and improved the essential amino acids (EAAs) levels. In addition, Se activated the antioxidants enzymes included in AsA-GSH cycle (SOD, CAT, APX, GR, DHAR, MDHAR, GSH, and AsA) and glyoxalase (Gly) system (Gly I and Gly II) and minimized the excessive generation of reactive oxygen species (ROS) and methylglyoxal (MG) contents in response to Cr stress. In a nutshell, Se (more effective at 5 µM) alleviated the Cr and MG induced phytotoxicity and oxidative damages by minimizing their (Cr and MG) accumulation and enhanced the plant growth, nutrients element level, nutrition quality by improving EAAs, antioxidant and Gly system. By considering the above-mentioned biomarkers, the addition of exogenous Se in Cr polluted soils might be effective approach to decrease the Cr uptake and its linked phytotoxicity in B. napus.

Ultrasonic seed treatment improved cadmium (Cd) tolerance in Brassica napus L.

Cadmium (Cd) affects crop growth and productivity by disrupting normal plant metabolism. To determinate whether ultrasonic (US) seed treatment can alleviate Cd stress in rape (Brassica napus L.), the seeds of two oilseed rape cultivars i.e., 'Youyanzao18' and 'Zaoshu104' were exposed to ultrasonic waves for 1 min at 20 KHz frequency. Seeds without US treatment were taken as control (CK). Results revealed that the germination rate of both cultivars was significantly (P < 0.05) higher in US treatment than CK only at 0 and 10 mg Cd L-1. The shoot and root length of both cultivars were significantly higher in US treatment than CK at all Cd treatments except the root length of Youyanzao18 at 50 mg Cd L-1. The fresh weight Youyanzao18 was significantly (P < 0.05) higher in US than CK except for Youyanzao18 at 25 mg Cd L-1. Moreover, the superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) activities and the proline, glutathione (GSH), and soluble protein contents in Youyanzao18 were relatively higher in the US treatment than CK. The malondialdehyde (MDA) contents were prominently reduced in US treatment than CK. The pods per plant, seeds per pod and rapeseed yield were increased by 15.9, 11.4, and 16.4% in Youyanzao18 and 10.3, 9.5, and 11.5% in Zaoshu104, respectively in US treatment, compared to CK. Moreover, the contents of Cd in root, stem, leaf, rape pod shell, and rapeseeds were comparatively less in US treatment than CK whereas the Cd concentrations in different plant parts of both rape cultivars were recorded as: leaf ˃ root ˃ stem ˃ rape pod shell ˃ rapeseed. In sum, the US treatment improved the morphological growth and rapeseed yield whereas reduced the Cd accumulation in different plant parts of rapeseed under Cd contaminated soil.

Proteomic changes in the xylem sap of Brassica napus under cadmium stress and functional validation.

BACKGROUND: The xylem sap of vascular plants primarily transports water and mineral nutrients from the roots to the shoots and also transports heavy metals such as cadmium (Cd). Proteomic changes in xylem sap is an important mechanism for detoxifying Cd by plants. However, it is unclear how proteins in xylem sap respond to Cd. Here, we investigated the effects of Cd stress on the xylem sap proteome of Brassica napus using a label-free shotgun proteomic approach to elucidate plant response mechanisms to Cd toxicity. RESULTS: We identified and quantified 672 proteins; 67% were predicted to be secretory, and 11% (73 proteins) were unique to Cd-treated samples. Cd stress caused statistically significant and biologically relevant abundance changes in 28 xylem sap proteins. Among these proteins, the metabolic pathways that were most affected were related to cell wall modifications, stress/oxidoreductases, and lipid and protein metabolism. We functionally validated a plant defensin-like protein, BnPDFL, which belongs to the stress/oxidoreductase category, that was unique to the Cd-treated samples and played a positive role in Cd tolerance. Subcellular localization analysis revealed that BnPDFL is cell wall-localized. In vitro Cd-binding assays revealed that BnPDFL has Cd-chelating activity. BnPDFL heterologous overexpression significantly enhanced Cd tolerance in E. coli and Arabidopsis. Functional disruption of Arabidopsis plant defensin genes AtPDF2.3 and AtPDF2.2, which are mainly expressed in root vascular bundles, significantly decreased Cd tolerance. CONCLUSIONS: Several xylem sap proteins in Brassica napus are differentially induced in response to Cd treatment, and plant defensin plays a positive role in Cd tolerance.

Balance between nitrogen use efficiency and cadmium tolerance in Brassica napus and Arabidopsis thaliana.

The transmembrane transport of NO3- and Cd2+ into plant cell vacuoles relies on the energy from their tonoplast proton pumps, V-ATPase and V-PPase. If the activity of these pumps is reduced, it results in less NO3- and Cd2+ being transported into the vacuoles, which contributes to better nitrogen use efficiency (NUE) and lower Cd2+ tolerance in plants. The physiological mechanisms that regulate the balance between NUE and Cd2+ tolerance remain unknown. In our study, two Brassica napus genotypes with differential NUEs, xiangyou 15 and 814, and Atclca-2 mutant and AtCAX4 over-expression line (AtCAX4-OE) of Arabidopsis thaliana, were used to investigate Cd2+ stress responses. We found that the Brassica napus genotype, with higher NUE, was more sensitive to Cd2+ stress. The AtCAX4-OE mutant, with higher Cd2+ vacuolar sequestration capacity (VSC), limited NO3- sequestration into root vacuoles and promoted NUE. Atclca-2 mutants, with decreased NO3- VSC, enhanced Cd2+ sequestration into root vacuoles and conferred greater Cd2+ tolerance than the WT. This may be due to the competition between Cd2+ andNO3- in the vacuoles for the energy provided by V-ATPase and V-PPase. Regulating the balance between Cd2+ and NO3- vacuolar accumulation by inhibiting the activity of CLCa transporter and increasing the activity of CAX4 transporter will simultaneously enhance both the NUE and Cd2+ tolerance of Brassica napus, essential for improving its Cd2+ phytoremediation potential.

Foliar-applied glutathione activates zinc transport from roots to shoots in oilseed rape.

Glutathione is a tripeptide involved in diverse aspects of plant metabolism. We investigated how the reduced form of glutathione, GSH, applied site-specifically to plants, affects zinc (Zn) distribution and behavior in oilseed rape plants (Brassica napus) cultured hydroponically. Foliar-applied GSH significantly increased the Zn content in shoots and the root-to-shoot Zn translocation ratio; furthermore, this treatment raised the Zn concentration in the cytosol of root cells and substantially enhanced Zn xylem loading. Notably, microarray analysis revealed that the gene encoding pectin methylesterase was upregulated in roots following foliar GSH treatment. We conclude that certain physiological signals triggered in response to foliar-applied GSH were transported via sieve tubes and functioned in root cells, which, in turn, increased Zn availability in roots by releasing Zn from their cell wall. Consequently, root-to-shoot translocation of Zn was activated and Zn accumulation in the shoot was markedly increased.