Studies On Brassinosteroid-induced Alleviation Of Polycyclic Aromatic Hydrocarbons Phytotoxicity And The Mechanism In Tomato

Polycyclic aromatic hydrocarbons (PAHs) are environmentally persistent organic micro pollutants. PAHs are of global environmental concerns because of their mutagenic, carcinogenic and teratogenic activities in humans. They are ubiquitous commonly found in soil, air and water, from where they enter plants, contaminate food chain and thus pose threat to human health. That is why, in planta detoxification of PAHs is very important to ensure food safety. Again, plants are important in removing PAHs, yet, understanding stress responses as well as strengthening plant tolerance and detoxification systems are important for plant based remediation of PAHs. Plant tolerance to PAHs is rate or capacity limiting as this is dependent on antioxidant and detoxification potential of plant. Hence, strengthening antioxidant and detoxification systems by hormonal supplementation may improve plant tolerance and degradation potential of xenobiotics like PAHs. Brassinosteroids (BRs), a class of plant-specific essential steroidal hormones are well known to induce plant tolerance against various abiotic stresses like high temperature, drought, salinity, heavy metal stress and also promote pesticide metabolism. Keeping in view the anti-stress properties of BRs, present study was carried out to understand the stress responses of tomato plant to PAHs (phenanthrene and pyrene), cadmium (Cd) and polychlorinated biphenyls (PCBs) as well as role of24-epibrassinolide (EBR) in stress amelioration through a series of experiments in different stages and conditions using morphological, physiological, biochemical, molecular and ultrastructural approaches. The salient findings are as follows:(1) The present experiment investigated the effects of seed treatment with various concentrations (0.01,1.0,100nM) of EBR on seed germination and early seedling growth in tomato under graded levels (30,100,300μM) of phenanthrene (PHE). Delayed and decreased seed germination; reduced length and fresh weight (FW) of shoot and root were observed following10days of PHE exposure in a dose dependent manner. However, seed treatment with EBR improved seed germination and increased length and FW of shoot and root. In addition, EBR remarkably restored the studied chlorophyll fluorescence parameters towards control levels. Different responses in antioxidant enzymes were observed following exposure to PHE, while malondialdehyde (MDA) was increased in a concentration dependent manner. Importantly, EBR pretreatment further increased activities of antioxidant enzymes but decreased the MDA content both in shoot and root of young tomato seedlings. Considering all the studied parameters, seed treatment with1.0nM EBR was most effective followed by100nM and0.01nM for improvement of germination and seedling growth under PHE stress in tomato.(2) A concentration-dependent decrease in growth, photosynthetic pigment contents, net photosynthetic rate (Pn), stomatal conductance (Gs), maximal quantum yield of PSII (Fv/Fm), effective quantum yield of PSⅡ (ΦPSⅡ) and photochemical quenching coefficient (qP) has been observed following phenanthrene (PHE) and pyrene (PYR) exposure (0,10,30,100and300μM). By contrast, non-photochemical quenching coefficient (NPQ) was increased. PHE was found to induce higher stress than PYR. However, foliar or root application of EBR (50nM and5nM, respectively) alleviated all those depressions with a sharp improvement in the activity of photosynthetic machinery. Superoxide dismutase (SOD) activity was gradually declined with increased concentration of PHE or PYR. However, the activities of guaiacol peroxidase (GPOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) as well as content of MDA were increased in a dose-dependent manner. In addition, both reduced glutathione (GSH) and oxidized glutathione (GSSG) were induced by PHE or PYR. Interestingly, EBR application in either form further increased enzymatic and non enzymatic antioxidants in tomato roots treated with PHE or PYR. Our results suggest that EBR has an anti-stress effect on tomato seedlings contaminated with PHE or PYR and this effect is mainly attributed by increased detoxification activity.(3) Exposure to PHE (300μM) for21d significantly decreased Pn which was attributed by both stomatal and nonstomatal factors. Changes in chlorophyll fluorescence parameters showed that PHE induced photoinhibition and subsequent damage to the photosynthetic machinery. Increased H2O2contents and localized H2O2accumulation in leaves indicated PHE-induced oxidative damage. All most all of the studied antioxidant enzymes were induced by PHE. Importantly, foliar application of EBR (0.1μM) significantly increased Pn and plant biomass over PHE alone. Furthermore, EBR+PHE showed remarkably decreased H2O2contents accompanied with increased antioxidant enzymes activities over PHE alone. Enhanced expression of detoxification related genes CYP90b3, GSH1and GST1suggested a strong coordinated detoxification under EBR+PHE treatment. Increased GSH and decreased GSSG contents resulted high GSH/GSSG in EBL+PHE treatment. In agreement with gene expression, a concurrent increment of glutathione S-transferase (GST) activity in EBL+PHE treatment over PHE alone was observed. Increased GST activity might promote conjugation of PHE with GSH and thereby improving metabolism of PHE. Meanwhile, remarkably lower PHE residue in root under EBR+PHE treatment suggested possible PHE degradation in root. Our results indicate that EBR application could be an effective tool to enhance plant tolerance against PHE stress via in planta detoxification.(4) This experiment was carried out to investigate the effects of PHE and EBR on secondary metabolism and ultra structure in tomato root. Exposure to PHE increased the activity of secondary metabolism related enzymes activities viz. GST, glucose-6-phosphate dehydrogenase (G6PDH), shikimate dehydrogenase (SKDH), phenylalanine ammonialyase (PAL) and cinnamyl alcohol dehydrogenase (CAD). The expressions of related genes were also induced by PHE. Meanwhile, EBR alone or in combination with PHE increased the enzymes activity and gene expression significantly over control and PHE alone, respectively. Consistent with enzymes activities contents of phenols, flavonoids and activity of DPPH were induced by PHE, while EBR application further increased all those studied parameters. Obvious ultrastructural alterations characterized by irregular shaped nucleus, less mitochondria, thin cell wall and more vacuolation were observed in PHE-treated root tip cells. However, EBR application saved root cell from severe PHE-induced damage which was clearly observed by relatively thick cell wall, more mitochondria, endoplasmic reticulum and small size vacuole in EBR+PHE-treated root tip cells. These observations suggest that EBR regulates secondary metabolism in tomato which might enhance tolerance to PHE.(5) Environmental pollution by heavy metal is often occurred with organic contaminants such as PAHs. However, plant stress physiology under co-contamination is poorly understood area. But knowledge on stress responses and stress tolerance is very important to develop and strengthen plant based remediation of mixed pollution as well as for food safety. Therefore, a greenhouse experiment was conducted to understand the stress responses of PHE and/or Cd to tomato plant and role of EBR in stress amelioration. PHE or Cd alone or in combination significantly reduced the biomass, Pn and photosynthetic pigment contents in tomato plant after40d of respective treatments. Surprisingly, combination of PHE and Cd did not reduce the growth synergistically, rather slightly improved the dry weight, Pn and leaf pigments. Single or dual pollution increased H2O2, MDA content, antioxidant enzymes activity and related genes expression which indicate an occurrence of oxidative stress. In addition, PHE and/or Cd induced the secondary metabolism and detoxification related enzymes activity as well as glutathione contents in tomato leaves. The foliar application of EBR on stressed plants, increased biomass, Pn, leaf pigments, but decreased H2O2and MDA contents by the action of enhanced antioxidant enzymes activity. EBR stimulates the secondary metabolism and xenobiotics detoxification related enzymes activity as well as gene expression towards enhanced tolerance to pollutant stress. Additionally, PHE and/or Cd residues were significantly decreased both in leaves and roots after application of EBR that indicate a possible promotion in degradation and detoxification of pollutants. Our results suggest that EBR could alleviate PHE-Cd co-contamination-induced stress by enhancing photosynthesis, antioxidant defense, secondary metabolism and pollutant detoxification capacity.(6) Cultivated vegetable species are continuously exposed to atmospheric PAHs; yet, information on their effects on different physiological parameters and related stress responses is an important part of stress biology. Two weeks foliar exposure of PHE resulted more or less dose dependent decrease in growth, photosynthesis and chlorophyll contents in five studied vegetables. With few exceptions, activities of antioxidant enzymes were upregulated following PHE exposure. Dose dependent increase in MDA contents together with H2O2accumulation suggested an occurrence of oxidative stress by PHE. However, to some extent, growth and antioxidant defense responses differ from species to species. Difference in defense capacity might result different tolerance and phytotoxicity among the studied vegetables. Taken together, phytotoxicity of PHE to five vegetables could be sequenced in the following order: pakchoi>cucumber>lettuce>tomato>chinese flowering cabbage.(7) PCBs are persistent organic pollutants often found in the atmosphere. Phytoremediation of airborne PCBs is an emerging new concept to minimize potential human exposure. However, effects of atmospheric PCBs on plant growth, photosynthesis and antioxidant defence system are poorly understood area. Meanwhile, BRs have been proposed as potential phytohormone for "Assisted phytoremediation by plant growth regulators" program. Hence, we studied the effects of PCBs and EBR on biomass accumulation, photo synthetic machinery and antioxidant system in tomato plants. PCBs (0.4,2.0and10μg/1) mist spray significantly decreased dry weight, photosynthesis, chlorophyll contents in a dose dependent manner. Both stomatal and non stomatal factors are involved in PCBs-induced photosynthetic inhibition. Meanwhile, Fv/Fm, ΦPSⅡ and qP were increasingly decreased by various levels of PCBs, suggested an induction of photoinhibition. Increased accumulation of H2O2accompanied with high lipid peroxidation suggested that seedlings faced oxidative stress upon PCBs exposure. Accordingly, antioxidant enzyme activity was inhibited due to inactivation of enzymes by over production of reactive oxygen species (ROS). In contrast, EBR (100nM) application increased biomass, photosynthetic capacity, chlorophyll contents and alleviated photoinhibition by enhancing Fv/Fm, ΦPSⅡ and qP. EBR significantly decreased harmful ROS accumulation and lipid peroxidation through upregulating antioxidant enzymes activity. Our results suggest a protective role of EBR against PCBs stress which may strengthen phytoremediation approaches by enhancing plant tolerance.