Species richness and phytoremediation potential of mine wastelands-native trees across the Zambian Copperbelt Region.

Mining activities are among the key sources of soil metal contamination in the Zambian Copperbelt, resulting in drastic landscape transformation. Plant species growing naturally on mine wastelands represent an asset for remediation on the disturbed ecosystems in the region. However, little is known about the suitability of Zambian native tree and shrub species for phytoremediation. The current study was carried to determine tree species richness and abundance on seven mine wastelands across the Zambian Copperbelt and evaluate their phytoremediation potential. Field inventory and post-hoc ecological analyses allowed identification of 32 native tree species, belonging to 13 different families, of which Fabaceae (34%) and Combretaceae (19%) predominated. Most of the identified tree species were found to be Cu, Co, Cr, Ni and Mo excluders. Among them, Rhus longipes (Anacardiaceae), Syzygium guineense (Myrtaceae), Senegalia polyacantha (Fabaceae) and Ficus craterostoma (Moraceae) were revealed as the most dominant tree species across the studied tailing dams (TDs) making them ideal candidates for metal phytostabilization. And coincidentally, their richness was positively correlated with high soil Cu concentration, a sought-after trait for phytoremediation of heavily polluted environment. Intriguingly, most identified tree species proved not suited for phytostabilization of Mn, Zn, B and Ba. On the other hand, species such as Annona senegalensis, Parinari curatellifolia, Dombeya rotundilifolia actively translocated these metals to leaves (TF > 1), indicating their potential for phytoextraction of Cu, Co, Cr, Ni, and Mo notably. Species richness and abundance significantly varied across the seven studied TDs. This was however barely influenced by soil metal contents, suggesting additional drivers dictating tree species-environment relationship in the context of studied TDs. The findings of this study provide crucial information in prospect of tree-based ecological restoration of mine wastelands, having revealed a diversified floristic composition of wastelands-native trees in the region, and clarified their respective phytoremediation attributes.

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.

Comprehensive proteomic analysis of arsenic induced toxicity reveals the mechanism of multilevel coordination of efficient defense and energy metabolism in two Brassica napus cultivars.

Arsenic (As) a non-essential element is of particular concern with respect to harmful effects on plant metabolism. While extensive studies have been conducted on the physiological responses of plants to increase As concentrations, however, molecular differences elucidating species-specific changes remain largely unknown. In the present experiment, two oilseed Brassica napus (B. napus) cultivars, ZS758 and ZD622, were treated by elevated As concentration. Their responses to the As stress have been investigated through pulse amplitude modulated fluorometer and isobaric tags based proteomic (iTRAQ) analysis. The chlorophyll fluorescence attributes showed that As stress significantly decrease the photochemical efficiency of photosystem II (PSII) and photosystem I (PSI) as well as the comparatively closed stomata observed under scanning electron microscopy (SEM). In this study, 65 proteins displayed increased abundance and 52 down-regulated were found in the control vs As comparison in cultivar ZS758, while 44 up and 67 down-regulated proteins were found in the control vs As comparison in ZD622. Metabolic pathways, followed by ribosome and biosynthesis of secondary metabolites were the dominant functional annotation categories among the differentially expressed protein (DEPs). Many genes involved in primary metabolism, stress and defense were found to be As-responsive DEPs and/or DEPs between these two cultivars. Based on these results, a schematic description of key processes involved in As tolerance in ZS758 and ZD622 is proposed, which suggests that higher tolerance in ZS758 depends on a multilevel coordination of efficient defense and energy metabolism. Real-time quantitative PCR supported the expression patterns of several genes encoding a protein similar to their corresponding DEPs. In addition, these findings could shed light in unraveling the molecular mechanisms of B. napus exposed to As stress and provide or improve essential understandings in the development of advanced B. napus cultivars against As resistance.

Ursolic Acid Limits Salt-Induced Oxidative Damage by Interfering With Nitric Oxide Production and Oxidative Defense Machinery in Rice.

Crops frequently encounter abiotic stresses, and salinity is a prime factor that suppresses plant growth and crop productivity, globally. Ursolic acid (UA) is a potential signaling molecule that alters physiology and biochemical processes and activates the defense mechanism in numerous animal models; however, effects of UA in plants under stress conditions and the underlying mechanism of stress alleviation have not been explored yet. This study examined the effects of foliar application of UA (100 µM) to mitigate salt stress in three rice cultivars (HZ, 712, and HAY). A pot experiment was conducted in a climate-controlled greenhouse with different salt stress treatments. The results indicated that exposure to NaCl-induced salinity reduces growth of rice cultivars by damaging chlorophyll pigment and chloroplast, particularly at a higher stress level. Application of UA alleviated adverse effects of salinity by suppressing oxidative stress (H2O2, O2-) and stimulating activities of enzymatic and non-enzymatic antioxidants (APX, CAT, POD, GR, GSH, AsA, proline, glycinebutane), as well as protecting cell membrane integrity (MDA, LOX, EL). Furthermore, UA application brought about a significant increase in the concentration of leaf nitric oxide (NO) by modulating the expression of NR and NOS enzymes. It seems that UA application also influenced Na+ efflux and maintained a lower cytosolic Na+/K+ ratio via concomitant upregulation of OsSOS1 and OsHKT1;5 in rice cultivars. The results of pharmacological tests have shown that supply of the NO scavenger (PTI) completely reversed the UA-induced salt tolerance in rice cultivars by quenching endogenous NO and triggering oxidative stress, Na+ uptake, and lipid peroxidation. The PTI application with UA and sodium nitroprusside (SNP) also caused growth retardation and a significant increase in Na+ uptake and oxidative stress in rice cultivars. This suggests that UA promoted salt tolerance of rice cultivars by triggering NO production and limiting toxic ion and reactive oxygen species (ROS) accumulation. These results revealed that both UA and NO are together required to develop a salt tolerance response in rice.

Synergistic effects of chromium and copper on photosynthetic inhibition, subcellular distribution, and related gene expression in Brassica napus cultivars.

Nowadays, modern plant physiology focuses on complex behavior of metal co-contaminants in agrosystems. Keeping this in view, the current study was conducted to investigate the response of two Brassica napus cultivars (Zheda 622 and ZS 758) under co-contamination of copper (Cu2+) and chromium (Cr6+) to observe their effects on plant growth, photosynthetic parameters, and subcellular distribution of these metals in leaves and roots. The results showed that exposure to Cu and Cr causes decline in plant growth, including biomass and plant height. Significant decrease in pigment concentration and the photosynthetic activity [photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (E), maximal quantum yield of photosystem II (Fv/Fm)] in leaves was also observed. Results of subcellular distribution of metals showed that Cu and Cr were predominantly distributed in cell wall and soluble fraction of roots and leaves. Moreover, Cu and Cr in cellular fractions showed a synergistic accumulation pattern under combined metal stress treatment. Both cultivars showed increased levels of reactive oxygen species (ROS), i.e., hydrogen peroxide (H2O2) and superoxide radical (O2•-), and significant modulation in the activities of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX)] under Cu/Cr alone or their combined treatments. Similarly, expression levels of defense-related genes, such as BnCat, BnApx, BnPrx, and BnSod, were also generally up-regulated compared with control. Electron micrographs (TEM) of the mesophyll and root tip cells indicated prominent alterations both in cellular and organelle levels. Additionally, Cr was found to be more toxic than Cu but less than their combined effect, as revealed by enhanced production of oxidative stress and a reduction in biomass production and photosynthetic activity. The present results also suggest that cultivar ZS 758 is more resistant to Cu/Cr than Zheda 622, due to better adapted metabolism and maintenance of structural integrity under metal stress.

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.

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.

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.

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.