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heavy metal toxicity and tolerance mechanism

1. HEAVY METAL TOXICITY AND TOLERANCE IN FIELD CROPS 1 Major Guide :- Dr. H. S. Bhadauriya Associate Professor Dep. of Genetics and Plant breeding Major Guide :- Dr. H. N. Zala Assistant Professor Dep. of Genetics and Plant breeding Prajapati Kinjalben Rameshbhai Reg. No. 04-AGRPH-02570-2021 Ph.D. (Plant Physiology)

2. OUTLINE 2 C onclusion

3. INTRODUCTION What is heavy metal ? Heavy metals are metallic element that have relatively high density usually greater than 5 g/cm 3 or their density is 4 to 5 times grater than the density of water Heavy metals are naturally occurring elements, which are widely distributed in the Earth’s crust ; they derive from rocks of volcanic, sedimentary or metamorphic origin, but in recent years, the occurrence of heavy metals in areas of agricultural and industrial activities has increased because of human activity. The rapid industrialization and poor management of industrial effluent is creating a more chance of heavy metal pollution. 3

4. Even at there exposure at minute level, heavy metal can have carcinogenic effect on human, animals and negative effect on soil microorganism and crop plants. Excessive concentration of heavy metal Viz , Cr, Cd, As, Se and Pb have been found in soils of agriculture land nearby cities, mines and industrial areas around the world. Among the various abiotic stresses , heavy metal stress is considered among the most noxious abiotic stress because it causes various damages at cellular, physiological and molecular levels. The presence of high heavy metals in the environment is a potential threat to the ecosystem and to human health. Heavy metals are one of the abiotic stresses contributing to high- yield losses of crops. ( Mariyam et al .) 4

5. Properties of heavy metals Heavy metal h ave high densities They are toxic in nature A major feature of heavy metal is non-degradability and persistence in all parts of the environment causing air, water and soil pollution . They occur near the bottom of the periodic table 5

6. Classification of heavy metal 6

7. Function of essential heavy metal Metals Function Copper Photosynthesis-Electron donor in photosystem I Zinc Many enzymes contain Zn-carbonic anhydrase, RNA polymerase Manganase Act as catalyst – involves in oxidation of carbohydrate Molybdenum Metabolism of N and S and biological N fixation Nickel Component of Urease enzyme Cobalt Useful in expansion of leaf disc, constituent of Vitamin B12 7

8. W hy there is need to study heavy metal in agriculture ? In terms of the degree of danger to the human population across the globe, heavy metals (HMs) are thought to be in second place among pollutants. In the last few decades, HMs have pulled ahead of pesticides and well-known pollutants such as carbon dioxide and sulfur dioxide. It is predicted that HMs may become the most dangerous contaminant, possibly surpassing solid and nuclear waste ( Lajayer et al . 2017). It has been reported that 70% of all HMs and their compounds routed in the human body came from food ( Jaishankar et al . 2014). HMs are released into the atmosphere, soil and water from a variety of sources and from anthropogenic activities ( Tchounwou et al . 2012). Later, they are introduced into the food chain and thus metal toxicity raises the risk and poses concerns for humans and animals. 8

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10. Natural sources The most important source of heavy metals is geologic parent material or rock outcroppings. The composition and concentration of heavy metals depend on the rock type and environmental conditions that activating the weathering process. The geologic plant materials generally have high concentrations of Cr, Mn , Co, Ni, Cu, Zn, Cd, Hg and Pb . However, class-wise the heavy metal concentrations vary within the rocks. Volcanoes have been reported to emit high levels of Al, Zn, Mn , Pb , Ni, Cu and Hg along with toxic and harmful gases (Seaward and Richardson, 1990). 10

11. Agriculture sources The inorganic and organic fertilizers are the most important sources of heavy metals to agricultural soil include liming, sewage sludge irrigation waters and pesticides. Others , particularly fungicides, inorganic fertilizers and phosphate fertilizers have variable levels of Cd, Cr, Ni, Pb and Zn depending on their sources. Cadmium is of particular concern in plants since it accumulates in leaves at very high levels, which may be consumed by animals or human being. Cadmium enrichment also occurs due to the application of sewage sludge, manure and limes. ( Yanqun et al., 2005 ). Although the levels of heavy metals in agricultural soil are very small, but repeated use of phosphate fertilizer and the long persistence time for metals, there may be dangerously high accumulation of some metals ( Verkleji 1993 ). Several heavy metal–based pesticides are used to control the diseases of grain, fruit crops and vegetables which are sources of heavy metal pollution to the soil ( Verkleji , 1993 ; Ross, 1994 ). 11

12. Table 1: Guideline for safe limit of heavy metal Metals Agricultural amendments Sewage sludge Compost refuse Farmyard manure Phosphate fertilizers Nitrate fertilizers Lime Pesticides Cr 8.40-600 1.8-410 1.1-55 66-245 3.2-19 10-15 - Ni 6-5,300 0.9-279 2.1-30 7-38 7-34 10-20 - Cu 50-8,000 13-3,580 2-172 1-300 - 2-125 - Zn 91-49,000 82-5,894 15-556 50-1,450 1-42 10-450 - Cd <1-3,410 0.01-100 0.1-0.8 0.1-190 0.05-8.5 0.04-0.1 - Pb 2-7,000 1.3-2,240 0.4-27 4-1,000 2-120 20-1,250 11-26 Sample Standards Cd Cu Pb Zn Ni Cr Indian standard ( Awashthi 2000) WHO/FAO (2007) 3-6 135-270 250-500 300-600 75-150 - Agri. soils (p g C I ) European union standards (EU 2002) 3 140 300 300 75 150 Table 2 : Heavy metal concentration ( μg /g ) in agricultural amendments Ross, 1994 12

13. Industrial sources Industrial sources of heavy metals include mining, refinement. For example, coalmines are sources of As, Cd, Fe, etc., which enrich the soil around the coalfield directly or indirectly. High temperature processing of metals such as smelting and castings emit metals in particulate and vapor forms. Vapor form of heavy metals such as As , Cd, Cu, Pb , Sn and Zn combine with water in the atmosphere to form aerosols. These may be either dispersed by wind (dry deposition) or precipitated in rainfall (wet deposition) causing contamination of soil or water bodies. Other industrial sources include processing of plastics, textiles, microelectronics, wood preservation and paper processing. Contamination of plants growing beneath the power line with high concentration of Cu is reported to be toxic to the grazing animals 13

14. Heavy metal contaminated sites in India 14 www.teriin.org Figure 1 : Location of the 320 probably contaminated sites in India ( MoEF&CC 2015) (No. of sites: Uttar Pradesh: 40, West Bengal: 36, Odisha: 31, Delhi: 28, Karnataka: 24, Gujarat: 23, Jharkhand: 14, Tamil Nadu: 13, Kerala: 11, Telangana : 9, and Punjab: 9) Cr Pb Hg As Cu Ranipet (TN) Ratlam (UP) Kodaikanal (TN) Tuticorin (TN) Tuticorin (TN) Vadodara (Gujarat) Vadodara (Gujarat) Ganjam (Orissa) Gangetic plain (WB) Malangkhand (MP) Talcher (Orissa) Korba ( Chattisgadh ) Singrauli (MP) Balia (UP) Kanpur (Up) Contamination of heavy metals in India has been observed across the nation. Nearly 718 districts have contaminated groundwater with arsenic, cadmium, chromium and lead (Mohan V 2018). Arsenic-contaminated groundwater covers major states such as Bihar, West Bengal, Uttar Pradesh, Jharkhand, Assam, Manipur and Chhattisgarh (WHO 2019). Ganga, the national river of India, is polluted with chromium, copper, nickel, lead and iron (Pandey, et al . 2019). Industrialization is one of the major contributors of contamination to sites like Vadodara (Gujarat), Ranipet (Tamil Nadu), Talcher (Orissa), Ratlam (Madhya Pradesh), Ganjam (Orissa), Singrauli (Madhya Pradesh), Balai (Uttar Pradesh) and Malanjkhand (Madhya Pradesh) ( Dotaniya and Jayanta 2016).

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16. 16 Fig. 2 Detrimental effect of heavy metal on plants Rattan et al . 2002

17. Fig. 3 Phytotoxic effects of heavy metals on plant 17

18. Plant heavy metal uptake mechanism Xylem unloading of heavy metal HM translocating through xylem Xylem loading of heavy metal xylem vessels in shoot heavy metal uptake in root Symplast pathway Apoplast pathway heavy metal translocate to shoots through symplast pathway Uptake pathway in root Uptake pathway in root Leaf cell vacuole Heavy metal enter into leaves through xylem unloading Storage of heavy metal into vacuoles Fig 4. M echanism of heavy metal uptake in plant 18

19. 19 HM-uptake have been reported via root system of the plants and also by foliar penetration. HMs transportation through soil matrix toward roots depend upon pH, organic matter, particle size, chemical nature of metal, cation -exchange capacity, micro flora etc. HM-ions are insoluble, unable to move by their own toward the vascular system of plants, so they form complexes for their free movement. Metal ions present in soil solution first gets adsorb onto root surface, followed by interaction with polysaccharides or carboxyl groups of root cells and mucilage uronic acids. Due to electrochemical gradient, plasma membrane (high negative potential) facilitates intake of metal ions. Heavy metal then follows two pathways in root cells, i.e., apoplastic and sym -plastic pathways via xylem loading. Various metal transporters, membrane transport proteins, chelators have been recognized which play significant role in transportation of metals toward aerial parts

20. (A) Level of heavy metal in fruits and vegetable Govind et al ., 2022 New Delhi, India 20 Heavy metal accumulation in fruits and vegetables and human health risk assessment

21. (B) Hazard index of toxic element for different vegetables New Delhi Govind et al . 21 (C) Metal pollution index in different vegetables and fruits

22. Heavy metal stress undesirably affects all phases of the plant from seed germination to the full growth of the plant, eventually, decreasing the overall yield of the economically vital crops. Sowing of seeds in soil that is excessively contaminated with toxic heavy metals results in declined germination, lowered growth of roots and shoots, fewer plant seedlings, and reduced biomass production. Metals are necessary for plant growth, but when they are present in excess amount, it causes severe toxicity and hindered the growth of the plant. Plants developed various mechanisms to survive the heavy metal stress effectively. Heavy metals can induce oxidative stress in plants, leading to damage to cellular components. Enhancing the antioxidant defense systems of plants can help mitigate this damage. This can be achieved by increasing the activity of enzymes such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), or by increasing the levels of non-enzymatic antioxidants such as glutathione and ascorbate . 22

23. Immobilization Cell wall compartmentalization Heavy metal chelation Antioxidant defense system Phytohormone Phytochelation Metallothioneins Heavy metal tolerance mechanism in crop Heavy metal tolerance mechanism in field crop 23

24. Physiological approaches for mitigating heavy metal toxicity in field crops 24

25. 1. Seed priming is considered as the most important instant approach to mitigate the adversarial effects of heavy metal stresses on plants as reported in different studies. Seed priming is denoted as a physiological strategy of seed hydration used to improve the metabolic process in plants to fasten the rate of germination, growth of plant seedlings as well as crop yield under both biotic and abiotic stress conditions. Seed priming upholds a momentary balance of ROS scavengers to alleviate the oxidative stress produced under stressful conditions. It results in the decreased production of hydrogen peroxide and malondialdehyde and improves the concentration of proline . Plants accumulate inactive signalling proteins in primed seeds. These inactive proteins become active soon after sensing the stressful conditions. Priming boost the vitality of the plant seed. Different priming agents are used in this technique like salicylic acid (SA) which is actively involved in the regulation of various physiological changes under stressful conditions 25

26. Fig 5. Seed priming with plant growth regulators Fig 6. Mechanism of seed priming to mitigate heavy metal toxicity 26

27. 27 HMS Plant species Priming agent References Cd Black Cumin ( Nigella sativa ) salicylic acid (SA) Espanany et al . (2016) Nano - ZnO Rice ( Oryza sativa) polyethylene glycol Sheteiwy et al . (2016) Cd Lettuce ( Lactuca sativa L.) salicylic acid Šabanović et al . (2018) Cd Lettuce Silicon Pereira et al . 2021 Pb Wheat ( Triticum aestivum ) Purslane extract, Aqueous chard extract Sobhy et al . (2019) Cd Maize ( Zea mays) Multiwall carbon nanotubes (MWCNTs) Chen et al . (2021) Cd Faba bean ( Vicia faba ) Calcium chloride Nouairi et al . (2019) Mn Sunflower (Helianthus annuus L.) Sulfur Nanoparticles Ragab & Saad-Allah (2020) Cd Cucumbers ( Cucumis sativus ) 3- epibrassinolide Shah et al . (2020) Cr Rice ( Oryza sativa) Brassinosteroids Basit et al . (2021) Cd Coriandrum sativum Karrikinolide Sardar et al . (2021) Cd Cowpea ( Vigna unguiculata (L.) Proline and Glycine Betaine Sadeghipour , (2020) Cd Wheat ( Triticum ) Salicylic acid Gul et al . (2020) Table 3 . Seed priming with different agents for mitigation of heavy metal stress

28. Plant growth regulators (PGRs) are known as synthetic and naturally occurring compounds that directly affect the all-important metabolic processes and development in higher plants, generally at small doses. Plant growth regulators directly affect the hormonal status of the plants, and are not phytotoxic. They don’t possess any nutritive value for the plant ( Rademacher , 2015). Plant growth regulators has the capacity to govern the majority of plant growth parameters from seed germination to reproduction and finally to plant death. Implementation of PGRs approach for the remediation of heavy metal stress is a very effective tool as reported in previous studies 28

29. Fig 7. Mechanism of phytohormone tolerance under heavy metal stress condition 29

30. HMS Plants PGR ( Phytohormone ) Mitigation Effects by PGRs References Co Grey mangrove ( Avicennia marina) Jasmonic acid (1, 10 μM ) JA efficiently reduced the accumulation of Cd in leaves. Yan et al. (2015) Cd Faba bean ( Vicia faba L.) Jasmonic acid (JA) JA alleviate the undesirable impacts of Cd stress by preventing the accumulation of Cd Ahmad et al . (2017) Pb Brassica campestris Salicylic acid (SA) Application of SA improve the growth and yield of the plant by regulating the antioxidant defense system Hasanuzzaman et al . (2019)   Cd Lettuce ( Lactuca sativa L.)   Abscisic acid (ABA) exogenous ABA increases the biomass, photosynthesis and activities of antioxidant enzymes under stressful conditions Dawuda et al . (2020) Cd Maize ( Zea mays L.) Salicylic acid (SA) Exogenous SA improved the process of photosynthesis and reduce the oxidative damaged occurred under Cd stress El Dakak and Hassan (2020) Pb Triticum aestivum L. Salicylic acid (SA) SA application considerably reduced the effect of Pb and improve the amount of biochemical traits of T. aestivum L. Gillani et al (2021) Cd Bean ( Phaseolus vulgaris L.) Salicylic acid (SA) Foliar applıcatıon of SA allevıate cd- encouraged ROS, methylglyoxal along with lipid peroxidation Hediji et al . (2021) Cd Vigna radiata L Gibberellins (GAs) Application GA improve the plant metabolism, enhance the photosynthetic pigments under Cd stress Hakla et al . (2021) 30 Table 4 . Representative studies on the role of plant growth regulators on different plants exposed to heavy metal stress

31. Fig. Mechanism of organic acid tolerance under heavy metal stress condition 31

32. Fig 8. Tolerance mechanism 32

33. 33 z Iran z Roya et al ., 2020 Pre- sowing seed treatment with salicylic acid and sodium hydrosulfide confers Pb toxicity tolerance in maize ( Zea mays L . ) Seeds were primed in 0.5 mM SA and 0.5 mM NaHS (sodium hydrosulfide ) individually and in combination of SA+NaHS with 0.25 mM SA and 0.25 mM NaHS for 12 h. The control seeds did not receive any pretreatment. After 12 h, seeds were germinated on moisturized filter paper for 3 days. Thereafter, seedlings were grown in pots and transferred to growth chamber under controlled conditions After 6 days, maize plants subjected to two Pb concentrations: 1- watered with Pb (NO 3 ) 2 (0 mM ), 2- watered with Pb (NO 3 ) 2 (2.5 mM ). Plants were harvested 9 days after Pb imposition. 1

34. Effects of seed priming with salicylic acid and sodium hydrosulfide on roots phenotype under stress conditions in maize plants under Pb stress 34 Fig. 8. Effects of SA, NaHS and SA+NaHS on roots phenotype under stress conditions. (A) Scale bar: 4 cm, (B) Scale bar: 4 cm, (C) Scale bar: 5 cm, (D) Scale bar: 4 cm, (E) Scale bar: 5 cm.

35. Effects of seed priming with salicylic acid and sodium hydrosulfide on growth parameters and total chlorophyll content in shoots and roots of maize plants under Pb stress 35

36. Effects of seed priming with salicylic acid and sodium hydrosulfide on Pb content and Fe content in shoots and roots of maize plants under Pb stress 36 58 % 66 % 70 % 63% 70 % 73 %

37. Effects of seed priming with salicylic acid and sodium hydrosulfide on cysteine and methionine content in shoots and roots of maize plants under Pb stress 37

38. 38 Effects of seed priming with salicylic acid and sodium hydrosulfide on MG and GlyI content in shoots and roots of maize plants under Pb stress

39. 39 Effects of seed priming with salicylic acid and sodium hydrosulfide on ZmSAMS , ZmGly1 (roots and shoots) expression of maize plants under Pb stress

40. Synergistic effect of 24-epibrassinolide and salicylic acid on photosynthetic efficiency and gene expression in Brassica juncea L. under Pb stress 40 The seeds were surface-sterilized with 0.01% HgCl 2 for 2 min followed by rinsing 5 times with distilled water . The surface-sterilized seeds were then presoaked in three different hormonal solutions including 10 –7 M EBL, 1 mM SA and a combination of EBL + SA for 8 h. These were then sown in earthen pots. Before sowing the seeds , treatment with different concentrations of lead ( 0.25 mM , 0.50 mM , and 0.75 mM , was given to soil. The plants were allowed to grow in earthen pots under natural conditions (5–10 °C) and they were harvested after 30, 60, and 90 days. Kohli et al., 2017 India 2

41. Synergistic effect of 24-epibrassinolide and salicylic acid on root length and shoot length under Pb stress in Brassica juncea L. Fig. (A) Effect of different concentrations of Pb (0.25 mM , 0.50 mM , and 0.75 mM ), EBL (10 –7 M), and SA (1 mM ) and their combinations on root length and shoot length of 30, 60 and 90-day-old plants of Brassica juncea L. 41

42. Synergistic effect of 24-epibrassinolide and salicylic acid on total chlorophyll and carotenoid content under Pb stress in Brassica juncea L Fig. (B) Effect of different concentrations of Pb (0.25 mM , 0.50 mM , and 0.75 mM ), EBL (10–7 M), and SA (1 mM ) and their combinations on total chlorophyll and carotenoid content of 30-, 60-, and 90-day-old plants of Brassica juncea L. 42

43. Synergistic effect of 24-epibrassinolide and salicylic acid on anthocyanin content under Pb stress in Brassica juncea L Fig. (C) Effect of different concentrations of Pb (0.25 mM , 0.50 mM , and 0.75 mM ), EBL (10 –7 M), and SA (1 mM ) and their combinations on anthocyanin content of 30-, 60-, and 90-day-old plants of Brassica juncea L. 43

44. Treatment Net photosynthetic rate (µ mol m 2 s –1 ) ( mean± SD) Stomatal conductance ( mmol CO 2 m 2 s –1 ) (mean ± SD) Pb EBL SA 30 DAS 60 DAS 90 DAS 30 DAS 60 DAS 90 DAS 13.23 ± 0.252 22.83 ± 0.702 38.23 ± 0.153 0.305 ± 0.004 0.504 ± 0.003 0.416 ± 0.005 10 –7 M 14.33 ± 0.252 29.53 ± 0.351 45.90 ± 0.361 0.320 ± 0.002 0.534 ± 0.003 0.455 ± 0.005 1 mM 13.97 ± 0.208 25.90 ± 0.265 42.13 ± 0.569 0.308 ± 0.003 0.528 ± 0.006 0.432 ± 0.003 10 –7 M 1 mM 16.63 ± 0.306 37.83 ± 0.503 53.47 ± 0.603 0.327 ± 0.004 0.552 ± 0.004 0.495 ± 0.004 0.25 mM 8.87 ± 0.351 21.60 ± 0.361 33.63 ± 0.231 0.247 ± 0.003 0.490 ± 0.005 0.393 ± 0.002 0.25 mM 10 –7 M 12.23 ± 0.252 25.13 ± 0.451 42.33 ± 0.961 0.274 ± 0.002 0.525 ± 0.004 0.427 ± 0.006 0.25 mM 1 mM 11.47 ± 0.351 24.03 ± 0.603 35.03 ± 0.503 0.272 ± 0.003 0.515 ± 0.004 0.409 ± 0.001 0.25 mM 10 –7 M 1 mM 13.43 ± 0.306 27.37 ± 0.611 48.10 ± 0.854 0.292 ± 0.004 0.550 ± 0.003 0.393 ± 0.002 0.50 mM 6.70 ± 0.200 18.10 ± 0.400 26.73 ± 0.473 0.198 ± 0.004 0.425 ± 0.003 0.323 ± 0.002 0.50 mM 10 –7 M 9.63 ± 0.404 22.60 ± 0.656 35.20 ± 0.361 0.251 ± 0.003 0.475 ± 0.005 0.374 ± 0.003 0.50 mM 1 mM 8.50 ± 0.300 19.00 ± 0.458 31.03 ± 0.416 0.233 ± 0.003 0.454 ± 0.004 0.359 ± 0.004 0.50 mM 10 –7 M 1 mM 10.43 ± 0.208 12.83 ± 0.611 39.70 ± 0.721 0.355 ± 0.004 0.487 ± 0.002 0.386 ± 0.005 0.75 mM 4.90 ± 0.361 15.53 ± 0.351 20.83 ± 0.306 0.155 ± 0.001 0.416 ± 0.005 0.312 ± 0.004 0.75 mM 10 –7 M 7.80 ± 0.265 18.03 ± 0.611 29.33 ± 0.208 0.205 ± 0.004 0.434 ± 0.003 0.336 ± 0.005 0.75 mM 1 mM 7.47 ± 0.321 15.53 ± 0.351 26.00 ± 0.794 0.195 ± 0.003 0.429 ± 0.004 0.327 ± 0.004 0.75 mM 10 –7 M 1 mM 9.53 ± 0.351 21.37 ± 0.758 34.43 ± 0.833 0.297 ± 0.003 0.416 ± 0.005 0.356 ± 0.004 F-ratio ( df 3, 32) treatment 1324.9** 1042.9** 2103.7** 2465.2** 1710.5** 2725.4** F-ratio ( df 3, 32) dose 384.5** 563.3** 1365.7** 1768.4** 314.8** 643.3** F-ratio ( df 9, 32) treatment × dose 7.95** 38.06** 10.60** 239.40** 11.33** 16.85** HSD 0.904 1.673 1.727 0.009 0.012 0.011 44 Table 3. Synergistic effect of 24-epibrassinolide (EBL) and salicylic acid (SA) on Net photosynthetic rate and Stomatal conductance under Pb stress in Brassica juncea L

45. Treatment Cellular CO 2 concentration (µ mol mol –1 ) (mean ± SD) Transpiration rate ( mmol m 2 s –1 ) (mean ± SD) Pb EBL SA 30 DAS 60 DAS 90 DAS 30 DAS 60 DAS 90 DAS 304 ± 2.5 444 ± 3.0 273 ± 2.0 1.18 ± 0.061 2.10 ± 0.051 3.95 ± 0.132 10 –7 M 327 ± 5.8 463 ± 2.5 294 ± 3.512 1.39 ± 0.025 2.35 ± 0.035 4.44 ± 0.099 1 mM 316 ± 5.6 456 ± 4.6 290 ± 4.5 1.29 ± 0.060 2.23 ± 0.085 4.17 ± 0.092 10 –7 M 1 mM 354± 3.5 478 ± 4.0 305 ± 4.0 1.59 ± 0.035 2.58 ± 0.040 4.88 ± 0.070 0.25 mM 255 ± 3.5 405 ± 3.5 246 ± 7.5 0.85 ± 0.003 1.54 ± 0.042 3.27 ± 0.057 0.25 mM 10 –7 M 295 ± 4.0 426 ± 2.1 275 ± 2.5 0.88 ± 0.024 1.74 ± 0.050 3.75 ± 0.04 0.25 mM 1 mM 284 ± 3.5 424 ± 3.5 265 ± 2.5 0.88 ± 0.027 1.66 ± 0.114 3.68 ± 0.070 0.25 mM 10 –7 M 1 mM 305 ± 4.0 435 ± 2.1 285 ± 3.5 0.91 ± 0.004 1.84 ± 0.076 3.86 ± 0.046 0.50 mM 242 ± 4.0 395 ± 5.0 225 ± 4.0 0.74 ± 0.006 0.99 ± 0.009 2.91 ± 0.066 0.50 mM 10 –7 M 275 ± 3.0 415 ± 4.0 254 ± 3.1 0.80 ± 0.006 1.23 ± 0.072 3.50 ± 0.207 0.50 mM 1 mM 264± 4.0 402 ± 2.1 245 ± 3.0 0.76 ± 0.009 1.18 ± 0.023 3.29 ± 0.045 0.50 mM 10 –7 M 1 mM 271± 3.1 425 ± 3.0 272 ± 3.5 0.82 ± 0.012 1.33 ± 0.060 3.77 ± 0.057 0.75 mM 217 ± 1.8 354 ± 3.5 211 ± 3.5 0.49 ± 0.010 0.73 ± 0.008 1.91 ± 0.091 0.75 mM 10 –7 M 247 ± 2.0 382 ± 2.1 232 ± 2.5 0.58 ± 0.017 0.88 ± 0.018 2.63 ± 0.104 0.75 mM 1 mM 236 ± 5.0 378 ± 3.0 234 ± 3.5 0.55 ± 0.035 0.86 ± 0.018 2.46 ± 0.123 0.75 mM 10 –7 M 1 mM 263 ± 4.0 393 ± 7.6 284 ± 3.5 0.62 ± 0.006 1.05 ± 0.046 3.45 ± 0.183 F-ratio ( df 3, 32) treatment 1017.9** 991.2** 396.3** 1756.9** 1998.8** 616.1** F-ratio ( df 3, 32) dose 265.9** 161.2** 308.2** 79.0** 117.3** 178.6** F-ratio ( df 9, 32) treatment × dose 8.66** 2.50* 16.64** 18.72** 3.99** 14.6** HSD 11.864 11.496 11.751 0.085 0.146 0.315 45 Table 4. Synergistic effect of 24-epibrassinolide and salicylic acid (SA) on total phenol and flavonoid content under Pb stress in Brassica juncea L

46. Treatment Total phenolic content (mg g –1 of DW) (mean ± SD) Flavonoid content (mg g –1 of DW) (mean ± SD) Pb EBL SA 30 DAS 60 DAS 90 DAS 30 DAS 60 DAS 90 DAS 10.41 ± 0.373 12.91 ± 0.151 14.32 ± 0.979 0.525 ± 0.013 0.543 ± 0.085 0.722 ± 0.014 10 –7 M 13.63 ± 0.072 18.83 ± 0.180 18.99 ± 0.355 0.812 ± 0.012 0.847 ± 0.041 1.016 ± 0.076 1 mM 10.84 ± 0.256 16.11 ± 0.284 16.99 ± 0.087 0.762 ± 0.090 0.639 ± 0.010 0.908 ± 0.050 10 –7 M 1 mM 18.68 ± 0.219 20.89 ± 0.259 21.50 ± 0.293 1.108 ± 0.018 0.561 ± 0.035 1.289 ± 0.018 0.25 mM 11.26 ± 0.155 18.16 ± 0.175 15.87 ± 0.222 0.660 ± 0.02 0.726 ± 0.135 0.689 ± 0.019 0.25 mM 10 –7 M 16.92 ± 0.154 24.58 ± 0.276 18.46 ± 0.155 0.791 ± 0.009 0.900 ± 0.049 1.303 ± 0.026 0.25 mM 1 mM 13.81 ± 0.137 20.69 ± 0.615 16.76 ± 0.196 0.764 ± 0.031 0.666 ± 0.019 0.914 ± 0.070 0.25 mM 10 –7 M 1 mM 18.17 ± 0.098 26.31 ± 0.103 21.52 ± 0.123 0.919 ± 0.156 0.948 ± 0.011 1.189 ± 0.010 0.50 mM 12.43 ± 0.154 20.35 ± 0.083 17.59 ± 0.231 0.893 ± 0.015 0.890 ± 0.079 0.729 ± 0.062 0.50 mM 10 –7 M 16.35 ± 0.393 23.41 ± 0.118 21.69 ± 0.193 1.075 ± 0.011 1.034 ± 0.009 1.258 ± 0.023 0.50 mM 1 mM 13.41 ± 0.221 21.99 ± 0.154 19.16 ± 0.226 0.960 ± 0.046 0.923 ± 0.106 1.084 ± 0.023 0.50 mM 10 –7 M 1 mM 19.55 ± 0.264 26.75 ± 0.143 26.35 ± 0.165 1.748 ± 0.012 1.204 ± 0.016 1.513 ± 0.014 0.75 mM 11.06 ± 0.190 23.47 ± 0.196 16.50 ± 0.136 1.063 ± 0.007 0.842 ± 0.084 1.009 ± 0.089 0.75 mM 10 –7 M 16.34 ± 0.738 28.29 ± 0.122 22.88 ± 0.180 1.263 ± 0.013 1.221 ± 0.050 1.533 ± 0.061 0.75 mM 1 mM 13.50 ± 0.150 25.21 ± 0.154 20.43 ± 0.262 1.141 ± 0.011 1.015 ± 0.049 1.167 ± 0.029 0.75 mM 10 –7 M 1 mM 19.28 ± 0.190 30.36 ± 0.257 31.05 ± 0.761 1.898 ± 0.622 1.318 ± 0.011 2.112 ± 0.008 F-ratio( df 3, 32) treatment 135.4** 2643.7** 768.8** 34.7** 86.6** 277.5** F-ratio( df 3, 32) dose 1974.1** 2019.2** 2122.1** 34.5** 86.4** 620.6** F-ratio( df 9, 32) treatment × dose 32.27** 101.27** 106.39** 2.43* 2.64* 36.62** HSD 0.819 0.720 0.873 0.491 0.187 0.136 46 Table 5. Synergistic effect of 24-epibrassinolide and salicylic acid (SA) on total phenol and flavonoid content under Pb stress in Brassica juncea L

47. Synergistic effect of 24-epibrassinolide and salicylic acid on gene expression of CHLASE and PSY under Pb stress in Brassica juncea L Figure 9. Effect of different concentrations of Pb (0.25 mM , 0.50 mM , and 0.75 mM ) and combination of EBL (10 –7 M) and SA (1 mM ) on gene expression analysis of CHLASE and PSY genes of 30-day-old plants of Brassica juncea L. 47

48. Synergistic effect of 24-epibrassinolide and salicylic acid on gene expression of CHS and PAL under Pb stress in Brassica juncea L Figure 10. Effect of different concentrations of Pb (0.25 mM , 0.50 mM , and 0.75 mM ) and combination of EBL (10 –7 M) and SA (1 mM ) on gene expression analysis of CHS and PAL genes of 30-day-old plants of Brassica juncea L 48

49. Effect of foliar spray of Zn combined with organic matters on the leaf weight, plant height and grain yield under Cd stress in rice Fig 11. Effects of the foliar application of Zn fertilizers on leaf weight ( A ), plant height ( B ), and grain yield ( C ). Zn-Lys, Zn–lysine ; Zn-AA, Zn–amino acid ; Zn-FA, Zn– fulvic acid; Zn + GSH, Zn combined with glutathione z Qinhui et al ., 2024 z China Control Zn-Lys Zn-AA Zn-FA Zn + GSH Control Zn-Lys Zn-AA Zn-FA Zn + GSH Control Zn-Lys Zn-AA Zn-FA Zn + GSH 49 3

50. Effect of foliar spray of Zn combined with organic matters on Cd bioaccumulation in grain under Cd stress in rice Zn-Lys , Zn–lysine; Zn-AA, Zn–amino acid; Zn-FA, Zn– fulvic acid;Zn + GSH, Zn combined with glutathione z Qinhui et al ., 2024 z China 56 % 81 % 68 % 69 % Cd concentration in grain mg/kg Control Zn-Lys Zn-AA Zn-FA Zn + GSH China food safety limit value 50

51. Effect of foliar spray of Zn combined with organic matters on Cd bioaccumulation in root, stem and leaves under Cd stress in rice Effects of the foliar application of Zn fertilizers on Cd concentrations in the roots, stems and leaves. Zn-Lys , Zn–lysine; Zn-AA, Zn–amino acid; Zn-FA, Zn– fulvic acid ; Zn + GSH, Zn combined with glutathione z Qinhui et al ., 2024 z China 41 % 40 % 51

52. Effect of foliar spray of Zn combined with organic matters on Cd translocation factors from the stems to grains under Cd stress in rice Effects of the foliar application of Zn fertilizers on Cd translocation factors from the stems to grains ( A ), leaves to grains ( B ), roots to grains ( C ), and roots to leaves ( D ). Zn-Lys , Zn–lysine; Zn-AA, Zn– amino acid; Zn-FA, Zn– fulvic acid ; Zn + GSH, Zn combined with glutathione Grain/stem Grain/leaf Grain/root Leaf/root Control Control Zn-Lys Zn-Lys Zn-AA Zn-AA Zn-FA Zn-FA Zn + GSH Zn + GSH Zn + GSH Control Control Zn-Lys Zn-Lys Zn-AA Zn-AA Zn-FA 52 Zn-FA Zn + GSH (A) (B) (C) (D)

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54. z India control IAA Cd Cd + IAA TIBA Cd + TIBA Cd + TIBA+IAA 54 Effect of foliar spray of IAA on Cd accumulation under Cd stress in tomato Cd content (µg/g) Cd content (µg/g) Root Shoot (A) (B)

55. Effect of foliar spray of IAA on growth parameters under Cd stress in tomato z Khan et al., 2019 z India Root length (cm/plant) control IAA Cd IAA Cd + IAA TIBA Cd + TIBA Cd + TIBA+IAA control control control IAA IAA IAA Cd Cd Cd Cd + IAA Cd + IAA Cd + IAA TIBA TIBA TIBA Cd + TIBA Cd + TIBA Cd + TIBA Cd + TIBA+IAA Cd + TIBA+IAA Cd + TIBA+IAA 55 S hoot length (cm/plant) Root fresh weight (mg/plant) Shoot fresh weight (mg/plant) (A) (B) (C) (D)

56. z India 56 Effect of foliar spray of IAA on SOR, H 2 O 2 , MDA and electric leakage under Cd stress in tomato SOR content ( nmol /g) H 2 O 2 content ( nmol /g) Electrolyte leakage (%) LPO (MDA equivelant ) (A) (B) (C) (D)

57. 57 Effect of foliar spray of IAA on APX, GR, DHAR and DHAR activity under Cd stress in tomato APX activity (U/mg) GR activity (U/mg) DHAR activity (U/mg) MDHAR activity (U/mg) (A) (B) (C) (D)