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.

Cell-Based Phenotyping Reveals QTL for Membrane Potential Maintenance Associated with Hypoxia and Salinity Stress Tolerance in Barley.

Waterlogging and salinity are two major abiotic stresses that hamper crop production world-wide resulting in multibillion losses. Plant abiotic stress tolerance is conferred by many interrelated mechanisms. Amongst these, the cell's ability to maintain membrane potential (MP) is considered to be amongst the most crucial traits, a positive relationship between the ability of plants to maintain highly negative MP and its tolerance to both salinity and waterlogging stress. However, no attempts have been made to identify quantitative trait loci (QTL) conferring this trait. In this study, the microelectrode MIFE technique was used to measure the plasma membrane potential of epidermal root cells of 150 double haploid (DH) lines of barley (Hordeum vulgare L.) from a cross between a Chinese landrace TX9425 and Japanese malting cultivar Naso Nijo under hypoxic conditions. A major QTL for the MP in the epidermal root cells in hypoxia-exposed plants was identified. This QTL was located on 2H, at a similar position to the QTL for waterlogging and salinity tolerance reported in previous studies. Further analysis confirmed that MP showed a significant contribution to both waterlogging and salinity tolerance. The fact that the QTL for MP was controlled by a single major QTL illustrates the power of the single-cell phenotyping approach and opens prospects for fine mapping this QTL and thus being more effective in marker assisted selection.

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.

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.

Hydrogen sulfide alleviates cadmium-induced morpho-physiological and ultrastructural changes in Brassica napus.

In the present study, role of hydrogen sulfide (H2S) in alleviating cadmium (Cd) induced stress in oilseed rape (Brassica napus L.) was studied under greenhouse conditions. Plants were grown hydroponically under three levels (0, 100, and 500µM) of Cd and three levels (0, 100 and 200µM) of H2S donor, sodium hydrosulfide (NaHS). Results showed that application of H2S significantly improved the plant growth, root morphology, chlorophyll contents, elements uptake and photosynthetic activity in B. napus plants under Cd stress. Moreover, addition of H2S reduced the Cd concentration in the leaves and roots of B. napus plants under Cd-toxicity. Exogenously applied H2S decreased the production of malondialdehyde and reactive oxygen species in the leaves and roots by improving the enzymatic antioxidant activities under Cd stress conditions. The microscopic examination indicated that application of exogenous H2S improved the cell structures and enabled a clean mesophyll cell having a well developed chloroplast with thylakoid membranes, and a number of mitochondria could be observed in the micrographs. A number of modifications could be found in root tip cell i.e. mature mitochondria, long endoplasmic reticulum and golgibodies under combined application of H2S and Cd. On the basis of these findings, it can be concluded that application of exogenous H2S has a protective role on plant growth, photosynthetic parameters, elements uptake, antioxidants enzyme activities and ultrastructural changes in B. napus under high Cd stress conditions.