Alleviation of Cadmium Stress in Wheat by Polyamines

CHAPTER 17

Alleviation of Cadmium Stress in Wheat by Polyamines Mostafa M. Rady1, Safia M.A. Ahmed1, Mohamed A. Seif El-Yazal1, Hanan A.A. Taie2 1Botany

Department, Faculty of Agriculture, Fayoum University, Fayoum, Egypt; 2Plant Biochemistry Department, National Research Centre, Cairo, Egypt

1. INTRODUCTION 1.1 Importance of the Studied Crop Wheat (Triticum aestivum L.), in the family Poaceae, is one of the most important cereal crops and the most widely grown for the feeding of humans and animals. In Egypt, wheat grains are used principally as human food, as is the case in most countries worldwide. Wheat provides 37% of the total calories and 40% of the protein in Egyptian diets (Zaki et al., 2007). Recently, a great focus of several Egyptian investigators has been directed toward increasing the productivity of wheat to minimize the gap between production and consumption by increasing the land area unit and cultivated area. Increases in wheat productivity per unit area can be achieved by breeding high-yield varieties and applying optimum cultural practices (Dewdar et al., 2008).

1.2 Cadmium Sources and Its Effects on Plants Abiotic stresses, including metal toxicity, have been shown not only to decrease wheat growth and productivity (Pandey et al., 2017) but also to cause great losses in the agricultural sector generally. Environmental pollution by heavy metals has increased because of manifold industrial activities, solid waste management, and agricultural improvements. The increased dependence of agriculture on chemical fertilizers, irrigation with sewage wastewater, and rapid industrialization are shown to add toxic metals to agricultural soils, causing harmful effects on the soil–plant environmental system. Cadmium (Cd) is considered a major environmental concern for the agricultural system, as its residence time in soil can be thousands of years (Kumar, 2013). Among the top toxins, Cd is ranked seventh. Concentrations of Cd found out in uncontaminated soils are Cadmium Tolerance in Plants: Agronomic, Molecular, Signaling, and Omic Approaches ISBN 978-0-12-815794-7 © 2019 Elsevier Inc. https://doi.org/10.1016/B978-0-12-815794-7.00017-5 All rights reserved.

463

464

Cadmium Tolerance in Plants: Agronomic, Molecular, Signaling, and Omic Approaches

usually below 0.5 mg kg−1 but can reach up to 3.0 mg kg−1 depending on soil parent materials (Nazar et al., 2012). Some phosphatic fertilizers and phosphorites contain high concentrations of Cd (4.77 mg kg−1) and are considered potential causes of increasing Cd contamination in rice (Khurana and Jhanji, 2014). Cd Accumulation in plants may cause several physiological, biochemical, and structural negative changes (Yan et al., 2015). Cd toxicity to plant cells is related to oxidative stress caused by overproduction of reactive oxygen species (ROS) (Yadav, 2010), stimulation or inhibition of antioxidant enzyme activities (Zheng et al., 2010), production of oxidative damage, and induction of lipid peroxidation (Rady and Hemida, 2015) and protein oxidation (Pena et al., 2006). On the other hand, carbohydrate metabolism (Gill and Tuteja, 2010a), amino acid and proline contents (Sharma and Dietz, 2006; Rady et al., 2016), and polyamine (PA) levels (Groppa and Benavides, 2008; Rady and Hemida, 2015) are altered. The sensitivity of plants to heavy metals depends on an interrelated network of physiological and molecular mechanisms such as (1) uptake and accumulation of metals through binding to extracellular exudates and cell wall constituents; (2) efflux of heavy metals from cytoplasm to extranuclear compartments including vacuoles; (3) complexation of heavy metal ions inside the cell by various substances—for example, organic and amino acids, phytochelatins (PCs), and metallothioneins; (4) accumulation of osmolytes and osmoprotectants, and induction of antioxidative enzymes; and (5) activation or modification of plant metabolism to allow adequate functioning of metabolic pathways and rapid repair of damaged cell structures (Cho et al., 2003).

1.3 Polyamines and Their Biosynthesis in Plants As a group of phytohormone-like aliphatic amine-protonated natural compounds, PAs are low-molecular-weight polycations found in all living organisms (Cohen, 1998) and are known to be essential for the growth and development of prokaryotes and eukaryotes (Tiburcio et al., 1990). They have been reported to be involved in many physiological processes including cell growth and development as well as plant stress responses (Gill and Tuteja, 2010b; Minocha et al., 2014). In plant cells, the diamine putrescine (PUT; NH2[CH2]4NH2), triamine spermidine (SPD; NH2[CH2]3NH[CH2]4NH2] and tetramine spermine [SPM; NH2[CH2]3NH[CH2]4NH[CH2]3NH2) constitute the major PAs. These three main PAs differ in the number of positive charges exhibited in the physiological pH of the cell. PUT synthesis proceeds through either

Alleviation of Cadmium Stress in Wheat by Polyamines

465

arginine decarboxylase (ADC) via agmatine (AGM) or ornithine decarboxylase (ODC), while SPD is synthesized by spermidine synthase through the addition to PUT of an aminopropyl moiety donated by decarboxylated S-adenosylmethionine formed by SAMDC. Conversion of AGM into PUT requires two distinct enzymes: N-carbamoylputrescine amidohydrolase and agmatine deiminase. SPD functions as a substrate for the synthesis of the higher-polyamine SPM. PUT is catabolized by diamine oxidases (DAOs) in a reaction that converts PUT into Δ1-pyrroline and generates ammonia and H2O2 as by-products (Gill and Tuteja, 2010b). DAOs are preferentially localized in plant cell walls, and hydrogen peroxide resulting from PUT catabolism may be important in lignifications and cross-linking reactions under normal and stress conditions. Following the oxidation of PUT, Δ1pyrroline is catabolized into γ-aminobutyric acid (Gill and Tuteja, 2010b), which is ultimately converted into succinic acid, a component of the Krebs cycle (Eller et al., 2006). PAs occur in the free form or as conjugates bound to phenolic acids and other low-molecular-weight compounds or to macromolecules such as proteins and nucleic acids. As such, they are found to stimulate DNA replication, transcription, and translation. They have been implicated in a wide range of biological processes in plant growth and development, including senescence, environmental stress, and infection pathogenesis. Therefore, PAs are implicated in a wide range of diverse environmental stresses (Rider et al., 2007; Yang et al., 2007; Kuznetsov and Shevyakova, 2010) including metal toxicity (Groppa and Benavides, 2008; Shevyakova et al., 2010; Rady and Hemida, 2015).

1.4 Exogenous Polyamine Application to Enhance Cadmium Stress Tolerance PAs are implicated in environmental abiotic stresses as a mechanism used by plants to increase stress tolerance as reported in work by Minocha et al. (2014). PA accumulations occur under abiotic stresses including heavy metals (Groppa and Benavides, 2008). Major changes in PA metabolism occur in response to various abiotic stress conditions (Bouchereau et al., 1999). Minocha et al. (2014) also noted that stress-induced PA accumulation is important in ameliorating plant responses to abiotic stress. Research on the effects of exogenous PA application on increased Cd tolerance in plants is quite scarce. Adopted by plants as a mechanism for tolerating stress, endogenous PAs are suggested to participate in sustaining plant cell membranes (e.g. membrane integrity; Borell et al., 1997), thus protecting them from damage under stress. But endogenous concentrations

466

Cadmium Tolerance in Plants: Agronomic, Molecular, Signaling, and Omic Approaches

of PAs are not enough to efficiently tolerate Cd stress. One way to increase endogenous PA content is to apply PA exogenously. As a support to plants, some evidence has suggested that exogenous application of PAs has been shown to stabilize plant cell membranes under Cd stress (Rady and Hemida, 2015; Rady et al., 2016). Foliar spray of SPM or SPD significantly increased the endogenous content of SPM and SPD and maintained membrane stability by reducing oxidative stress and promoting antioxidant enzyme activity in Cd-exposed Malus hupehensis (Zhao and Yang, 2008). Earlier, Weinstein et al. (1986) reported Cd2+ concentrations as low as 10 mM resulted in accumulated PUT in oat seedlings of up to 10-fold higher due to greater induction of ADC activities; however, SPD and SPM contents did not change or had negligible variation. In bean leaves, Cd2+ induced accumulation of PUT in free and soluble conjugated forms but not in the insoluble fraction.This suggests a rapid exchange between free-form and soluble conjugated forms of PUT. Further, Groppa et al. (2003) showed that accumulation of PUT in Cd2+-treated wheat leaves was mediated by a simultaneous enhancement of ADC and ODC activities, but only ODC was responsible for the increased level of PUT in Cu2+-treated leaf discs. In wheat leaves, the authors proved that increased PUT content under metal treatments could be due to an inhibition of DAO activity leading to a lower degradation of PUT and that this fact might contribute to the higher PUT content observed under Cd2+ treatment. However, Groppa et al. (2001) reported that Cd2+ at 0.5 mM reduced PUT content in sunflower leaf discs due to decreased ADC and ODC activities, even though DAO activity was significantly reduced. Cu also decreased ADC activity but did not modify ODC and DAO activities, and thus the decrease in ADC activity might be responsible for the reduced PUT levels observed. In relation to the antioxidative behavior of PAs in Cd2+-treated leaf discs, Groppa et al. (2001) further reported that the exogenous application of 1 Mm SPM was implicated in protection against metalinduced oxidation damage through the recovery of glutathione reductase (GR) and superoxide dismutase (SOD) activities and reduced the formation of thiobarbituric acid reactive substances (TBARS) and H2O2. The precise mechanism by which SPM exerts its antioxidant role is yet to be fully explored. Tang et al. (2005) demonstrated that exogenous application of SPD could reduce Cd2+-induced ROS generation depending on the level of Cd2+. The authors concluded that exogenous SPD effectively improved the tolerance of Typha latifolia to Cd2+ stress by elevating the activities of antioxidant enzymes, especially GR activity, directly or indirectly inhibiting ROS generation and reducing malondialdehyde production.

Alleviation of Cadmium Stress in Wheat by Polyamines

467

Cd-induced oxidative stress was alleviated by exogenous application of PAs in some Gramineae crops including wheat (Groppa et al., 2007), studying PAs as antioxidants in wheat leaves under Cd2+ stress. The oxidative damage produced by Cd2+ stress was evidenced by an increase in TBARS and decrease in glutathione and SPM content. Preapplication with SPM completely reverted the metal-induced TBARS increase, whereas metal-dependent H2O2 deposition on leaf segments was considerably reduced in SPM-pretreated leaf segments.These results suggest that SPM could be exerting a certain antioxidative function by protecting tissues from metal-induced oxidative damage. Exogenously applied PAs have been reported to reverse the effects of Cd in terms of oxidative stress by preventing ROS formation or removing metals (Groppa et al., 2001). SPM was found to regulate the stabilization of Cd-accrued DNA methylation (Kumar et al., 2012). It has been reported that SPD and SPM protected Cd-induced oxidative damage by blocking the uptake of Cd in plants (Hsu and Kao, 2007) and by inducing genes for PA metabolism, DAO, and SAMDC in Cd-exposed Glycine max (Chmielowska-Bąk et al., 2013). Moreover, exogenous application of SPD improved the tolerance of Typha by increasing the antioxidant system and inhibiting ROS generation under Cd stress (Tang et al., 2005). The protective effect of PUT preapplication in mung bean and of SPD and SPM pretreatments in rice were associated with increases in GSH and some other antioxidants (Nahar et al., 2016).

1.5 Aim of the Study Based on the preceding overview, it is hypothesized that exogenous application of PAs (i.e., SPM, SPD, and PUT) will enhance the growth and productivity of wheat plants grown under Cd stress. Thus, the main objective of the present study was to assess to what extent seed presoaking and plant foliage spraying with SPM, SPD, and PUT could improve growth and productivity, physiobiochemical attributes, and the activity of the antioxidative defense system (i.e., enzymatic and nonenzymatic antioxidants) in wheat plants grown under Cd stress.

2. MATERIALS AND METHODS 2.1 Plant Material, Growing Conditions, and Treatments Authorized seeds of wheat (Triticum aestivum L., cv. Sakha 94) were surface sterilized with a 1% sodium hypochlorite solution for 2 min, thoroughly washed several times with distilled water, and left to air-dry. After excluding a number of seeds for control (no treatment, seed soaking in PAs, spraying with PAs, or Cd

468

Cadmium Tolerance in Plants: Agronomic, Molecular, Signaling, and Omic Approaches

addition with irrigation) and the same number for treatment with only Cd added in irrigation solution, the sterilized seeds were divided into two groups; the first was soaked for 8 h at room temperature in 0.25 mM SPM, 0.50 mM SPD, or 1.0 mM PUT. The second group had not been soaked but were cultivated, and the seedlings were sprayed with 0.25 mM SPM, 0.50 mM SPD, or 1.0 mM PUT.After air-drying overnight, 20 PA-treated and PA-untreated seeds were sown on November 1, 2013, and December 1, 2014, in plastic pots (40cm diameter, 35-cm depth), each of which was filled prior with 12 kg sand. Sand used in this study was washed for each of the two seasons with commercial HCl (10% conc.) for 24 h to remove all anions and cations and was then washed with distilled water several times to remove excess acid. Pots (n = 160 for eight treatments; 20 pots for each) were arranged for growing plants in an open greenhouse at the Experimental Farm of the Faculty of Agriculture, Faiyum, Egypt.Two weeks after sowing (WAS), wheat seedlings in each pot of the second group were sprayed to runoff with different PAs at the mentioned concentrations; the sprays were repeated two times, at five and eight WAS. To ensure optimal penetration into leaf tissues, 0.1% (v/v) Tween-20 was added to foliar sprays as a surfactant. The selection of the tested PA concentrations for either seed soaking or seedling spray, and spray timing, were based on the best response in our preliminary studies (data not shown). Both presoaked and sprayed seedlings were irrigated with Cd2+-containing nutrient solution. Irrigation with Cd2+-containing nutrient solutions was started with the first foliar spray. The average day and night temperatures were 19 ± 3°C and 10 ± 2°C, respectively. The relative humidity ranged from 62.0% to 65.1%, and day-length ranged from 10 to 11 h. A half-strength Hoagland’s nutrient solution was used for the two-year experiments (Hoagland and Arnon, 1950). The Cd2+-free nutrient solution was supplied every 3 days to all pots up to complete emergence. Excess solution was drained through holes in the bases of the pots.At this stage (2 WAS), seedlings were thinned to 10 per pot, and 2 mM Cd2+ using CdCl2 was added to the half-strength Hoagland’s nutrient solution. Each pot was supplemented every 3 days with 1 L of Cd2+-containing Hoagland’s nutrient solution. The 2-mM Cd2+ dose was also selected based on our preliminary studies (data not shown).This Cd2+ dose greatly affected wheat seedling growth.The Cd2+ concentration in the medium was maintained at 2 mM using inductively coupled plasma atomic emission spectrometry (ICP- AES, IRIS-Advan type, Thermo, USA). Initial soil pH was 5.5, but it was corrected to 6.8 by adding 3 g of CaCO3 per pot. The pH of the nutrient solution was adjusted to 7.0 with diluted HCl or NaOH.The experimental layout was a completely randomized design with 20 replicates/pots for each of the eight treatments.The experiments were continued up to harvest, but the irrigation with Cd2+-containing nutrient

Alleviation of Cadmium Stress in Wheat by Polyamines

469

solution was terminated 72 days from sowing after exposing the seedlings to Cd2+ stress for 60 days/20 irrigations.The 75-day-old seedlings from each treatment were collected for growth and physiobiochemical measurements, while a yield component assessment was conducted at the harvest stage.

2.2 Assessment of Plant Growth, Yield Components, and Water Use Efficiency Sampling took place directly before the flowering stage to assess growth characteristics and at harvest to determine the yield and its components. At the sampling date (10 WAS), six pots of each treatment were randomly taken and plants were obtained carefully (a moderate stream of tap water was used to remove plants from surrounding soil) and cleaned of adhering dirt with tap and then distilled water. The plants were separated into two parts: shoots and roots. Plant height, root, and flag leaf lengths were measured using a meter scale.The numbers of tillers and leaves per plants were taken. Flag leaf area was assessed using a Planimeter Planix 7. Root system volume was measured using a gradual cylinder with water, where the difference between water surface in the cylinder after and before submerging a root indicates root volume. Each plant part was first weighed (fresh weights, or FWs, of shoots and roots), and subsequently dried in an electric oven at 70 ± 2°C until constant weight was achieved. The dried materials, after being weighed (dry weights, or DWs), were finally ground to a fine powder for chemical analyses. At harvest stage (22 WAS), spikes were collected to determine yield and its components as well as grain quality (grain Cd content). Spike length was measured using a meter scale. The numbers of spikelets and grains per spike were counted, and weights of 1000-grain and grains per plant were recorded. Water use efficiency (WUE) was calculated by dividing grain yield (g pot−1) by the total amount of water added to each pot. For grain yield,WUEG was calculated from grain yield according to Stanhill (1987) as follows: WUEG = Grain yield (g)/Total water used (L).

2.3 Assessment of Tissue Health and Leaf Photosynthetic Pigments Assessments of relative water content (Weatherly (1950) with modifications by Osman and Rady (2014)), membrane stability index (Premchandra et al. (1990) with modifications by Rady (2011)), and electrolyte leakage (Sullivan and Ross (1979)) using fresh fully expanded leaves excluding the midrib were completed.

470

Cadmium Tolerance in Plants: Agronomic, Molecular, Signaling, and Omic Approaches

Chlorophylls and total carotenoids were extracted by leaf homogenization in 80% acetone. Measures at 663, 646, and 470 nm were done, and pigment contents were calculated according to the formulae of Welburn and Lichtenthaler (1984): Chlorophyll “a” = 12.21 E663 − 2.81 E646 μg−1 Chlorophyll “b” = 20.13 E646 − 5.03 E663 μg−1 Total carotenoids = 1000 E470 − 3.27 chl. “a” − 104 chl. “b”/229 μg−1

2.4 Assays of Enzymatic Activities Catalase (CAT) activity was determined by the method of Beer and Sizer (1952) as follows. Fresh leaf (0.5 g) was homogenized in polytron with 4 mL phosphate buffer (pH 7.0). Extract was centrifuged at 400 rpm for 15 min. CAT activity was measured in the supernatant using 1.9 mL of reagentgrade water, 1.0 mL of H2O2 as substrate and 0.1 mL of extract, and expressed (nm min−1 g−1 fresh leaf) as changes in OD at 240 nm. Activity of SOD was assayed based on a SOD-inhibitable reduction of nitro blue tetrazolium chloride (NBT) by superoxide radicals under illumination in a growth chamber (Cakmak and Marschner, 1992). The reaction medium (5 mL) comprised 50 mM of phosphate buffer (pH 7.6), 0.1 mM sodium ethylenediaminetetraacetic acid, enzyme extracts (50–150  μL), 50 mM Na2CO3 (pH 10.2), 12 mM l-methionine, 75 μM p-NBT, and 2 μM riboflavin added in glass vials, and the reaction was initiated by turning the lights on at an intensity of about 700 μE m−2s−1 with the assay lasting 15 min. The amount of enzyme extract that caused a 50% decrease in the SODinhibitable NBT reduction was defined as a unit at 560 nm. Peroxidase (POD) activity was determined as outlined by Maehly and Chance (1954). Fresh leaf (0.5 g) was homogenized in polytron with 4 mL of phosphate buffer (pH 6.0). Extract was centrifuged at 400 rpm for 15 min. Enzyme activity was measured in the supernatant using a reaction mixture comprising 1.5 mL of phosphate buffer, 1.5 mL of H2O2 (20 volume), and 1.5 mL of 0.04 M catechol solution as substrate and 0.1 mL of extract, and expressed (nm min−1 g−1 fresh leaves) as changes in OD at 470 nm.

2.5 Determination of Macronutrients The wet digestion of 0.1 g of fine dried plant material was conducted using a sulfuric and perchloric acid mixture as mentioned by Piper (1947). Phosphorus (%) was colorimetrically determined using the chlorostannusmolybdophosphoric blue color method in a sulfuric acid system as described by Jackson (1967). Potassium (%) was determined using a Perkin–Elmer

Alleviation of Cadmium Stress in Wheat by Polyamines

471

flame photometer (Page et al., 1982). Nitrogen (%) was determined in powdery dried leaf by the orange–G dye colorimetric method of Hafez and Mikkelsen (1981).

2.6 Determination of Cadmium (Cd2+) Content and Its Translocation Index The powdery dried plant samples and grains were ashed at 500°C for 12 h to determine Cd concentration. The ashed samples were dissolved in 3.3% HNO3 (v/v). The concentration of Cd was measured by inductively coupled plasma optical emission spectroscopy (ICP-OES,Varian, Australia).The Cd measurements in plant materials were checked against certificated Cd values in different reference plant materials obtained from the National Institute of Standards and Technology (Gaithersburg, USA). The Cd content was calculated by multiplying the DW values of roots or shoots by their Cd concentration values. A sample of 10-mL nutrient solution from each pot was collected at different time intervals as indicated in the results section. Because water was absorbed by plants due to transpiration, before collecting sample solutions, each solution was filled to the initial volume to avoid any concentration effects due to reduced water levels in pots. The Cd concentrations in collected samples were measured by ICP-OES. The results were calculated in terms of root dry matter production, based on absorption of Cd in 1 h in micromoles (μmol Cd g−1 root DW h−1) and calculated as cumulative Cd absorption based on absorption of Cd in micromoles (μmol Cd g−1 root DW h−1).

2.7 Determination of Total Soluble Sugars and Free Proline Contents Soluble sugars were extracted from dried leaf with 80% ethanol. One gram of the dried tissue was homogenized with 80% ethanol and then put in a boiling water bath for 15 min. After cooling, the extract was filtered and the filtrate oven-dried at 60°C. It was then dissolved in a known volume of water to be ready for soluble sugar determination by the anthrone sulfuric acid method described by Scott and Melvin (1956). Free proline concentrations in shoots and roots were determined from 1 g of dried material according to Bates et al. (1973). Extraction was made with an aqueous solution of 3% sulfosalicylic acid, and the extract was reacted with ninhydrin acid and glacial acetic acid. Absorbance was read at 520 nm, and proline content was calculated per unit of DW.

472

Cadmium Tolerance in Plants: Agronomic, Molecular, Signaling, and Omic Approaches

2.8 Statistical Analysis Data were statistically analyzed by the technique of ANOVA for completely randomized design using MSTAT-C (Michigan, USA), and LSD at a 5% level of significance was used to test differences between treatment means.

3. RESULTS AND DISCUSSIONS 3.1 Effects of Seed Soaking and Foliar Spraying With Polyamines on the Growth Characteristics of CadmiumStressed Wheat Plants Data presented in Tables 17.1 and 17.2 show that the presence of Cd (2 mM) in the growth medium resulted in a marked decrease in growth traits (i.e., plant height, tillers per plant, leaves per plant, flag leaf length, flag leaf area per plant, root size, root length, shoot FW and DW, and root FW and DW) of wheat plants compared with controls (without any treatment). However, treating wheat plants subjected to Cd with SPM, SPD, and PUT, by seed soaking or foliar spraying, significantly improved the aforementioned growth characteristics. Generally, seed soaking treatments were found to be more effective than foliar spray treatments. In addition, it has been found that seed soaking in PUT was the best treatment under Cd stress. This treatment significantly increased plant height, tillers per plant, leaves per plant, flag leaf length, flag leaf area per plant, root system volume, root length, shoot FW and DW, and root FW and DW under Cd stress by 88.9%, 59.1%, 83.5%, 75.6%, 82.4%, 108.0%, 49.8%, 94.0%, 71.5%, 87.7%, and 254.0%, respectively compared with measurements for the Cd-stressed treatment during the 2013–14 season. In addition, these growth characteristics were increased by PUT application for seeds under Cd stress by 55.2%, 62.4%, 84.8%, 99.3%, 117.1%, 123.1%, 42.6%, 100.3%, 140.1%, 95.1%, and 266.0%, respectively compared with levels under Cd-stressed treatment during the 2014–15 season. Study results reveal that Cd is toxic, as reported earlier by Mohan and Hosetti (1999). Cd was found to reduce plant vigor and inhibit plant growth (Semida et al., 2015; Rady and Hemida, 2015). Kopittke et al. (2010) reported that a toxic concentration of Cd was 0.3 μM. Cd2+-induced growth inhibition proved to come from inhibiting cell division and cell elongation rate, which consequently resulted in declining plant biomass production. This mainly occurred due to an irreversible inhibition of the proton pump responsible for these processes (Choudhury and Panda, 2004). The reduction in wheat plant growth was associated with a Cd2+-induced increase in Cd2+ ion content (Table 17.3) and electrolyte leakage (Fig. 17.1),

Table 17.1  Effect of Polyamines on Growth Characteristics of Cadmium-Stressed Wheat Plants Treatments Parameters Soaking in PAs

Spraying With PAs

Plant Height (cm)

Tillers Number Plant−1

Leaves Number Plant−1

Flag Leaf Length (cm)

Flag Leaf Area plant−1 (cm2)

30.83 12.83 16.80 17.50 19.53 22.77 22.83 24.23 1.20

2.33 1.05 1.00 1.33 1.67 1.33 1.63 1.67 0.05

4.67 2.00 3.15 3.27 3.33 3.30 3.36 3.67 0.16

9.90 4.80 6.43 6.53 7.07 7.03 7.53 8.43 0.09

19.93 9.47 11.53 12.55 16.77 14.00 14.33 17.27 1.70

33.27 15.03 20.77 20.97 22.07 22.23 22.60 23.33 0.18

2.67 1.09 1.21 1.40 1.73 1.39 1.67 1.77 0.05

4.53 2.04 3.25 3.35 3.47 3.38 3.45 3.77 0.16

9.80 4.20 6.37 6.53 7.20 7.00 7.13 8.37 0.09

17.67 7.00 10.17 10.47 13.90 12.16 13.07 15.20 1.26

2013–14 Season

Cd + SPM Cd + SPD Cd + PUT – – –

2014–15 Season

Controla Cd – – – Cd + SPM Cd + SPD Cd + PUT LSD0.05

means plants without treatment; neither Cd nor PAs.

473

aControl

Cd + SPM Cd + SPD Cd + PUT – – –

Alleviation of Cadmium Stress in Wheat by Polyamines

Controla Cd – – – Cd + SPM Cd + SPD Cd + PUT LSD0.05

Spraying With PAs

Fresh Weight (g)

Dry Weight (g)

Root System Size (cm3)

Root Length (cm)

Shoots

Roots

Shoots

Roots

3.87 1.50 2.00 2.26 3.00 2. 67 2. 87 3.12 0.17

10.67 6.23 8.10 8.57 9.20 8.57 9.09 9.33 0.16

9.35 2.98 4.09 4.35 5.11 4.50 4.75 5.78 0.53

6.17 2.98 3.85 4.14 4.87 4.34 4.51 5.11 0.18

1.51 0.57 0.71 0.81 1.01 0.76 0.86 1.07 0.19

2.40 0.50 1.13 1.37 1.64 1.21 1.33 1.77 0.53

3.37 1.43 2.06 2.23 2.83 2.53 2.71 3.19 0.19

11.33 6.60 8.37 8.67 9.25 8.57 8.90 9.41 0.15

9.70 3.04 4.33 4.79 5.56 5.08 5.34 6.09 0.17

6.14 2.07 4.03 4.60 5.00 4.63 4.70 4.97 0.49

1.58 0.61 0.81 0.88 1.04 0.85 1.01 1.19 0.16

2.36 0.47 1.15 1.46 1.60 1.32 1.60 1.72 0.14

2013–14 Season

Controla Cd – – – Cd + SPM Cd + SPD Cd + PUT LSD0.05

Cd + SPM Cd + SPD Cd + PUT – – –

2014–15 Season

Controla Cd – – – Cd + SPM Cd + SPD Cd + PUT LSD0.05 aControl

Cd + SPM Cd + SPD Cd + PUT – – –

means plants without treatments; neither Cd nor PAs.

Cadmium Tolerance in Plants: Agronomic, Molecular, Signaling, and Omic Approaches

Soaking in PAs

474

Table 17.2  Effect of Polyamines on Growth Characteristics of Cadmium-Stressed Wheat Plants Treatments Parameters

Table 17.3  Effect of Polyamines on Nutrient and Cadmium Content in Cadmium-Stressed Wheat Plants Treatments Parameters Cd Soaking in PAs

Spraying With PAs

Content (mg g−1 DW) N (% DW)

P (% DW)

K (% DW)

Shoots

Roots

Translocation Index

1.98 1.77 1.80 1.83 1.93 1.84 1.88 1.95 0.04

0.38 0.14 0.25 0.29 0.21 0.30 0.33 0.26 0.03

3.39 1.68 2.98 3.08 2.83 3.12 3.24 2.91 0.17

0.17 19.98 17.13 11.81 16.82 16.26 11.33 15.85 1.23

0.18 38.56 34.91 29.74 33.55 34.58 29.22 33.14 1.30

33.33 19.92 10.86 8.95 12.14 10.68 9.53 11.2 7.71

2.00 1.79 1.83 1.86 1.96 1.85 1.89 1.98 0.04

0.36 0.15 0.24 0.29 0.21 0.27 0.32 0.25 0.03

3.50 1.66 2.98 3.16 2.89 3.14 3.31 2.98 0.24

0.19 22.40 18.43 12.48 17.75 17.90 12.19 16.86 1.23

0.19 40.45 35.19 31.62 34.48 34.58 29.21 32.12 1.30

33.33 23.69 13.09 9.06 12.8 11.8 10.36 13.75 7.69

2013–14 Season

Cd + SPM Cd + SPD Cd + PUT – – –

2014–15 Season

aControl

Cd + SPM Cd + SPD Cd + PUT – – –

means plants without treatments; neither Cd nor PAs.

475

Controla Cd – – – Cd + SPM Cd + SPD Cd + PUT LSD0.05

Alleviation of Cadmium Stress in Wheat by Polyamines

Controla Cd – – – Cd + SPM Cd + SPD Cd + PUT LSD0.05

476

Cadmium Tolerance in Plants: Agronomic, Molecular, Signaling, and Omic Approaches

Figure 17.1  Effect of polyamines on MSI, EL, and RWC of Cd-stressed wheat plants. *Control means plants without treatment, neither Cd nor PAs. LSD = MSI, 0.34 and 0.35; EL, 1.45 and 1.37; and RWC, 1.56 and 1.55; for two seasons, respectively.

Alleviation of Cadmium Stress in Wheat by Polyamines

477

Figure 17.2  Effect of polyamines on leaf photosynthetic pigments of Cd-stressed wheat plants. *Control means plants without treatment, neither Cd nor PAs. LSD = chlorophyll‒ a, 0.01 and 0.01; chlorophyll‒b, 0.02 and 0.02; and carotenoids, 0.01 and 0.01; for two seasons, respectively.

reductions in leaf photosynthetic pigments (Fig. 17.2), and disturbances in water relations and membrane stabilities (Fig. 17.1). These results indicate that wheat plants had reduced Cd2+ stress tolerance, particularly at the level of 2 mM Cd used in this study. This reduction in wheat Cd2+ stress tolerance may be attributed to the increased Cd2+ absorption by roots and its translocation to shoots in abundant amounts (Table 17.3). This negatively affects cell physiological and biochemical processes through Cd2+ oxidative damage to cellular components (Rady and Hemida, 2015).

478

Cadmium Tolerance in Plants: Agronomic, Molecular, Signaling, and Omic Approaches

To alleviate and repair the damage caused by ROS due to the presence of Cd in the plant growth medium, the exogenous application of antioxidants such as PAs enables plants to develop a complex antioxidant defense system to increase the cellular defense strategy against Cd2+-induced oxidative stress. Exogenous application of SPM, SPD, and PUT decreased the deleterious effects of Cd2+ and significantly enhanced wheat plant growth characteristics as shown in Tables 17.1 and 17.2. The improved growth of wheat plants grown under Cd2+ stress may be attributed to the reduced content of endogenous Cd2+ ions (Table 17.3). It has been reported that PAs are involved in various biochemical and physiological processes related to plant growth and development (Walden et al., 1997), which could explain the prevention role of SPM, SPD, and PUT in Cd-induced plant growth inhibition.

3.2 Effects of Seed Soaking and Foliar Spraying With Polyamines on the Membrane Stability Index, Relative Water Content, and Electrolyte Leakage of CadmiumStressed Wheat Plants Data in Fig. 17.1 show that the tissue health of Cd2+-treated plants in terms of MSI, RWC, and EL were significantly troubled compared with that of control plants over both growing seasons. However, treating wheat plants subjected to Cd with SPM, SPD, and PUT, either by seed soaking or by foliar spraying, significantly improved MSI and RWC. Generally, seed soaking treatments were found to be more effective than foliar spray treatments. In addition, it has been found that seed soaking in SPD was the best treatment under Cd stress. This treatment significantly increased MSI and RWC under Cd stress by 15.2% and 14.07%, respectively, compared with Cd-stressed treatment, during the 2013–14 season. In addition, MSI and RWC were increased by SPD application for seeds under Cd stress by 11.7% and 15.5%, respectively, compared with Cd-stressed treatment in 2014–15 season. In contrast, EL behavior reverses trend, decreasing from SPD application in seeds under Cd stress by 58.3% in the first season and by 54.2% in the second season compared with controls. MSI and RWC of wheat plants were significantly reduced, while EL was significantly increased, in the presence of Cd (Rady and Hemida, 2015).This result is mainly attributed to the injury occurred to plasma membranes. Metwali et al. (2013) have reported that Cd disrupted plant water relations, and its negative effect can be observed in the uptake, transport, and transpiration of water in plants. The integrity of cell membranes was assessed

Alleviation of Cadmium Stress in Wheat by Polyamines

479

indirectly by investigating solution conductivity, which illustrated leakage of electrolytes from cells and significantly increasing EL under Cd stress. This confirmed that Cd toxicity in wheat leaf was linked to free radical processes in membrane components leading to alterations in membrane stability and increasing their permeability (Ahmad et al., 2011). Cd, like any other transition metal, is known to bind to sulfhydryl groups and destabilize the membrane system by inducing the formation of disulfide links leading to distortion in the structure and function of membrane ion channels (Aravind and Prasad, 2005). De Maria et al. (2013) reported that Cd can alter water relations by disturbing the water balance through its effects on stomatal conductance, water transport, and cell wall elasticity, significantly reducing RWC under Cd stress. However, PAs modulated the Cd stress effect on RWC by reducing Cd2+ ion content and through their role in water integrity as illustrated by Kubis et al. (2014). The stimulation effect of stress on EL% might be attributed to injury of the plasma membrane. As active redox metals, heavy metals can induce overproduction of ROS, such as the hydrogen peroxide hydroxyl radical, and the superoxide anion directly, which in turn leads to lipid peroxidation and oxidative stress (Zhang et al., 2015). Heavy metal–caused oxidative damage was demonstrated by Vantová et al. (2013). In the present study, exogenous application of PAs reduced the adverse effects of Cd on wheat seedlings and maintained their MSI and RWC values at significant increased levels above those of Cd2+-stressed seedlings. Measurements of EL values in PUT-, SPD-, or SPM-treated plants showed a reverse trend for MSI and RWC (Rady and Hemida, 2015). The protective role of PAs in plant cells is due to their radical scavenging ability (Sharma and Dietz, 2006) and the inhibition of lipid peroxidation (Velikova et al., 2000). This protective role for PAs was confirmed by Groppa et al. (2001), Rhee et al. (2007), and Rady and Hemida (2015), thus proving the increased tolerance to Cd that results from exogenously supplied PAs.

3.3 Effects of Seed Soaking and Foliar Spraying With Polyamines on Leaf Photosynthetic Pigments of CadmiumStressed Wheat Plants The presence of Cd (2 mM) in the growth medium resulted in a marked decrease in the contents of leaf photosynthetic pigments (chlorophyll‒a, chlorophyll‒b and carotenoids) of wheat plants compared with controls (without any treatment) as shown in Fig. 17.2. However, treating wheat plants grown under Cd stress with SPM, SPD, and PUT, either by seed soaking or by foliar application, significantly improved the contents of leaf

480

Cadmium Tolerance in Plants: Agronomic, Molecular, Signaling, and Omic Approaches

photosynthetic pigments. In general, seed soaking was better than foliar spray under Cd stress. In addition, it has been found that seed soaking using SPD was the best treatment, generating the best values of chlorophylls and carotenoids under Cd stress. This treatment significantly increased chlorophyll‒a, chlorophyll‒b, and carotenoids under Cd stress by 75.0%, 84.6%, and 171.4%, respectively, compared with Cd-stressed treatment in the 2013–14 season. In addition, these pigments were increased by SPD application to seeds under Cd stress by 106.7%, 100.0%, and 175.0%, respectively, compared with Cd-stressed controls in the 2014–15 season. Photosynthesis inhibition is a well-documented response of plants to toxic metal ions (Deng et al., 2014; Ouyang et al., 2012; Zhang et al., 2015). Chlorophyll fluorescence was reduced by exposure to heavy metals as reported by Baumann et al. (2009). The authors documented that chlorophyll fluorescence was reduced significantly at 10 μmol mL−1 of Cd added to algal cultures in Fucus vesiculosus, Cladophora rupestris, Palmaria palmata, and Polysiphonia lanosa. In plants, the first visible symptom of Cd toxicity is chlorosis appearing on leaves (Das et al., 1997; Baryla et al., 2001). Cd has been found to damage the chloroplast structure (Rascio et al., 1993; Ouzounidou et al., 1997). The high Cd level in leaf tissues has been suggested as an indirect influence on chlorophyll content via metabolic disruption and premature senescence (Vassilev et al., 1997). Generally, chlorophyll‒b is more sensitive than chlorophyll‒a (Xiong et al., 2006). The mechanism of chlorophyll breakdown into phytol, Mg, and the primary cleavage product of the porphyrin ring occurs in four consecutive steps. This reaction is catalyzed by chlorophyllase, Mg-dechelatase, pheophorbide a oxygenase, and red chlorophyll catabolite reductase. Loss of typical chlorophyll green color occurs only after cleavage of the porphyrin ring (Harpaz-Saad et al., 2007).The observed decrease in photosynthetic activity is often a more sensitive measure than is pigment content. It was demonstrated also that plant treatment with PAs prevented loss in chlorophyll content, led to membrane stabilization, and delayed senescence (Borrell et al., 1997; Velikova et al., 2000). The exogenous application of PAs probably prevents chlorophyll loss, protecting the thylakoid membrane structure due to their cation characteristics. It is possible that exogenous PA applications induce physiological effects by means of a nonspecific influence on plasmalemma. SPD, as a best treatment, could regulate the structures and functions of the photosynthetic apparatus through its interaction with the thylakoid membrane (Yiu et al., 2009), which might be related to increased chlorophyll content in plants.

Alleviation of Cadmium Stress in Wheat by Polyamines

481

3.4 Effects of Seed Soaking and Foliar Spraying With Polyamines on Nutritional Status of Cadmium-Stressed Wheat Plants Data presented in Table 17.3 reveal the effects of seed soaking or foliar spray with different PAs on Cd-stressed plant nutrient contents. The presence of Cd at a 2-mM concentration in the growth medium resulted in significant reductions in the content of N, P, and K in plants compared with Cd-untreated controls. However, treating plants subjected to Cd stress with SPM, SPD, and PUT, either by seed soaking or foliar application, significantly improved the content of tested nutrients. Generally, seed soaking treatments were more effective than foliar sprays. In addition, it has been found that seed soaking in PUT (for N) or SPD (for P and K) was the best treatment under Cd stress. These treatments significantly increased N, P, and K under Cd stress by 10.5%, 116.1%, and 98.5%, respectively, compared with Cd-stressed controls in the 2013–14 season. In addition, these plant nutrients were increased by PUT (for N) or SPD (for P and K) application to seeds under Cd stress by 10.8%, 96.4%, and 102.2%, respectively, compared with Cd-stressed controls in the 2014–15 season. Cd inhibits nitrate reductase activity and consequently decreases nitrate absorption and transfer from roots to shoots (Hernandez-Lopez et al., 1996). In this study, it was observed that Cd treatment led to decreased N content in wheat plants.This result agreed with findings of Yildiz (2005) for tomato and corn. In general, heavy metal toxicity is related to heavy metal binding to enzymes and in such case results in changes in and inhibition of metabolism (Van Assche and Clijsters, 1990). Thus it was reported for different plants that Cd changed the enzyme activities included in nitrogen metabolism (Boussama et al., 1999). P content also decreased with Cd treatment. Similar decreased P content with Cd treatment has been reported for birch shoots (Gussarson and Jensen, 1992). The decreased P content with Cd treatments was due to the negative impacts of Cd on P nutrition and metabolism in plants.Thus Cd can inhibit activities of enzymes related to P metabolism (Sharma and Dubey, 2005). Cd generally decreased Ca and K content in both roots and shoots of in vitro plantlets (Gonçalves et al., 2009). Content of K in durum wheat seedlings were decreased with Cd treatments, which attributed to decreasing K uptakes by seedlings under Cd stress (Veselov et al., 2003). Significant decreases in the K content of white lupine (Lupinus albus) shoots, with 18 and 45 μM Cd treatments, were also reported (Zornoza et al., 2002). Such decreases in K concentrations may be related to ATPase, responsible for active K uptake (Lindberg and Wingstrand, 1985).

482

Cadmium Tolerance in Plants: Agronomic, Molecular, Signaling, and Omic Approaches

The application of PAs restored nutrients (N, P, and K), increasing their content in wheat plants. Among these increased mineral nutrients, K+ is considered an osmolyte suggested to participate in sustaining membrane integrity (Wang et al., 2013) and maintaining cells in a turgid state, and consequently reducing ion leakage from cells under Cd2+stress. Application of SPM, SPD, and PUT caused an increased P content. This increase suggests that PAs increased water uptake by the root system and consequently increased the passive uptake and translocation of phosphate ions from soil that were driven by the transpiration stream (Jalil et al., 1994).

3.5 Effects of Seed Soaking and Foliar Spraying With Polyamines on Cd2+ Content and Its Translocation Index in Cadmium-Stressed Wheat Plants Data shown in Table 17.3 exhibit that Cd2+-treated wheat plants showed significant increases in Cd2+ content but exhibited significant reductions in the Cd translocation index compared with untreated control plants over both the 2013–14 and 2014–15 seasons. Under Cd2+ stress, SPM-, SPD-, and PUT-pretreated plants showed significant decreases in Cd2+ content and further decreases in the Cd translocation index. Among all PAs, SPD was the best treatment, being the most effective in preventing the absorption and translocation of Cd. From data shown in Table 17.3, Cd content and its translocation index are inversely correlated. Cd translocation index values were significantly reduced with PA applications, both seed soaking and foliar spray.There is no special transport channel for a nonessential element such as Cd within plants. Nonessential metal elements are transported into plants via transporters and channels for essential elements such as Ca and K (Clemens et al., 1998). Although Cd is not required as an essential nutrient in higher plants, the bioaccumulation index of Cd may exceed that of other elements (Kabata-Pendias and Pendias, 1992). Wheat plants accumulated higher amounts of Cd in roots than in shoots as shown in Table 17.3. Such a correlation was observed in the case of Cd. Fecenko et al. (1997) recorded the highest Cd accumulation in barley roots, with lower accumulation in straw, and the lowest in grain, in an experiment to determine Cd uptake and localization in spring barley. A significantly higher concentration of Cd in barley roots than in straw was observed (Tlustos et al., 1997). Root growth is more sensitive to heavy metals than shoot growth is. This evidence correlated with data that had significantly lower root length under Cd treatments compared with controls.

Alleviation of Cadmium Stress in Wheat by Polyamines

483

The ameliorating effect of PAs on heavy metals by inducing synthesis of specific proteins may be attributed to the role of PAs in increasing the tolerance of wheat plants to heavy metals (Walters, 2003), particularly Cd. The exogenous application of PAs plays an important role in increasing the tolerance of wheat plants to Cd treatment by decreasing the accumulation of Cd contents in roots and consequently in shoots as compared with their corresponding controls. This repairing effect induced by exogenous PAs may occur because PAs (1) increase PC production, particularly in roots; (2) increase cell wall and vacuolar storage of these heavy metals; (3) increase detoxification of heavy metals by increasing the accumulation of these metals in the trichomes of the leaves and peduncles of wheat plants (Alsokari and Aldesuquy, 2011); and (4) act as efficient antioxidants and free radical scavengers under these stresses and increase root exudates into soil (biosphere).

3.6 Effects of Seed Soaking and Foliar Spraying With Polyamines on the Osmoprotectant Contents of CadmiumStressed Wheat Plants Data shown in Table 17.4 exhibit that Cd2+-treated plants showed significant increases in the content of soluble sugars and free proline compared with corresponding untreated control plants over two studied seasons. Under Cd2+ stress, SPM-, SPD-, and PUT-pretreated plants showed significant increases in soluble sugars and free proline contents compared with corresponding PA-untreated plants over both growing seasons. It has been found, generally, that seed soaking in PUT (for soluble sugars) or SPD (for free proline) was the best treatment under Cd stress. PUT, the best treatment for soluble sugars, showed a significant reduction in total soluble sugars under Cd stress by 6.3% in the 2013–14 season and 5.7% during 2014–15. SPD treatment was the best for free proline content, since it significantly increased free proline by 50.8% in the 2013–14 season and 44.6% during 2014–15. Exposure to heavy metals is often accomplished by synthesis and accumulation of various metabolites, including some amino acids, particularly free proline and soluble carbohydrates (Jha and Dubey, 2004), in response to metal stress such as Cd2+ stress that can be elucidated by the degradation of certain proteins and/or by de novo synthesis of amino acids (Sharma and Dietz, 2006). Free proline, as an amino acid, is one of the most common metabolites synthesized in response to stress as part of a general adaptation syndrome to unfavorable environmental conditions. The accumulation of

Spraying With PAs

Soluble Sugars (mg g−1 DW)

Free Proline (mg g−1 FW)

SOD (Units g−1 FW)

POD (nm min−1 g−1)

CAT (nm min−1 g−1)

48.5 73.6 54.0 57.9 65.5 59.0 62.9 68.6 3.5

0.45 0.60 0.77 0.83 0.72 0.81 0.84 0.78 0.06

0.15 0.22 0.07 0.18 0.11 0.10 0.21 0.13 0.02

0.36 0.19 0.57 0.52 0.55 0.57 0.60 0.53 0.06

0.20 0.16 0.26 0.25 0.25 0.24 0.24 0.27 0.04

49.2 71.3 53.0 57.0 63.0 58.8 62.7 68.3 3.5

0.52 0.65 0.74 0.78 0.71 0.89 0.85 0.81 0.07

0.19 0.18 0.30 0.29 0.29 0.49 0.51 0.50 0.02

0.28 0.12 0.25 0.25 0.25 0.25 0.25 0.27 0.05

0.16 0.18 0.30 0.29 0.29 0.49 0.51 0.50 0.04

2013–14 Season

Controla Cd – – – Cd + SPM Cd + SPD Cd + PUT LSD0.05

Cd + SPM Cd + SPD Cd + PUT – – –

2014–15 Season

Controla Cd – – – Cd + SPM Cd + SPD Cd + PUT LSD0.05 aControl

Cd + SPM Cd + SPD Cd + PUT – – –

means plants without treatments; neither Cd nor PAs.

Cadmium Tolerance in Plants: Agronomic, Molecular, Signaling, and Omic Approaches

Soaking in PAs

484

Table 17.4  Effect of Polyamines on Osmoprotectants and Enzyme Activities in Cadmium-Stressed Wheat Plants Treatments Parameters

Alleviation of Cadmium Stress in Wheat by Polyamines

485

fluid compatible osmoregulation, chelation and detoxification of metals, protection of enzymes, and regulation of cytosolic acidity are the functions of proline that stabilize the machinery of protein synthesis and the trapping of ROS (Sharmila and Pardha Saradhi, 2002). Many studies have shown that PAs are involved in defense mechanisms during biotic and abiotic stresses (Rhee et al., 2007; Groppa and Benavides, 2008).The increase in proline as a result of Cd2+stress is noticed in the present study; however, the application of SPM, SPD, and PUT reduced the endogenous Cd concentration in wheat plants, showing additional mechanisms by which these PAs reduce the adverse effects of Cd2+ stress. El Bassiouny et al. (2008) found that application of PA treatment had a favorable effect on the synthesis and accumulation of carbohydrates in the leaves of wheat plants. In most cases, plants sprayed with PAs at different concentrations had a higher total carbohydrate content in their leaves compared with the untreated control.With the application of SPD, leaves accumulated more compatible osmolytes such as soluble sugars (i.e., glucose and fructose), sugar alcohol, sorbitol, and proline in response to stress (Szepesi, 2006).

3.7 Effects of Seed Soaking and Foliar Spraying With Polyamines on the Enzymatic Antioxidant Activity of Cadmium-Stressed Wheat Plants Data presented in Table 17.4 show that the presence of Cd in the growth medium resulted in significant decreases in the activities of POD and CAT, which resulted in significantly increased SOD activity in wheat plants compared with controls (without any treatment). However, treating wheat plants subjected to Cd stress with SPM, SPD, and PUT, either by seed soaking or foliar spraying, significantly improved the activities of the aforementioned enzymes, except for the activity of SOD, which fluctuated in increased, decreased, or constant state. Generally, enzyme activity data cannot identify whether one treatment or another is best. These results are parallel for both growing seasons. It has been found that seed soaking in PUT was the best for CAT, and seed soaking in SPD was best for SOD and POD, in both the 2013–14 and 2014–15 seasons. Antioxidant enzymes are very good biochemical markers of stress, and increasing their activities could potentially alleviate oxidative stress induced by Cd stress and aid external supports such as antioxidants. Under Cd stress, PAs induced improved activities of all enzymes tested in this study, which may be attributed to the antioxidative characteristics of PAs against the generation of ROS, and plants try to force this by stimulation of the antioxidant defense

486

Cadmium Tolerance in Plants: Agronomic, Molecular, Signaling, and Omic Approaches

system (Li et al., 2011). Overproduction of ROS is a common consequence of different stress factors including Cd. To maintain metabolic functions under stress conditions, a balance between generation and degradation of ROS is required; otherwise, oxidative injuries may occur. The level of ROS in plant tissues is controlled by an antioxidant system that consists of antioxidant enzymes, including SOD, CAT, and POD, and nonenzymatic low-molecularweight antioxidants including total soluble sugars and proline (Schutzendubel and Polle, 2002). Therefore, it was expected that exposure of wheat plants to Cd stress would enhance the activities of antioxidant enzymes. Therefore, improved values of enzyme activities were recorded in plants subjected to simultaneous Cd stress with the application of SPM, SPD, and PUT, either by seed soaking or by foliar spraying.The aforementioned enzymatic components play a relevant role in mitigating heavy metal stress, including Cd stress. Several studies have revealed that treatment with heavy metal enhances ROS formation, and thus substantial increases in the activities of SOD, CAT, and ascorbate peroxidase were observed (Bashri and Prasad, 2015). The enzyme SOD is the first line of defense to counter the superoxide (O2−%) radical. It catalyzes the conversion of O2%− to H2O2 that is subsequently converted to H2O by the enzyme POD (Alscher et al., 2002).This explanation elucidates that only SOD activity was increased under Cd stress before PA treatment. CAT scavenges H2O2 by converting it to H2O and finally O2, and POD reduces H2O2 using several reductants, such as ascorbate, guaiacol, and phenolic compounds (Apel and Hirt, 2004). Several mechanisms have been suggested to explain the increased oxidant resistance attributed to PAs; e.g. PAs could act as direct radical scavengers (Drolet et al., 1986).They could bind to antioxidant enzymes or be conjugated to antioxidant molecules and allow them to permeate to the sites of oxidative stress (Poduslo and Curran, 1996), or may interact with membranes stabilizing molecular complexes of thylakoid membranes (Besford et al., 1993). Plasma membrane function may be rapidly affected by heavy metals (Quartacci et al., 2001). PA application resulted in increased enzymatic activity as a reported synergy between antioxidant enzymes and PAs in a protective mechanism (Gill and Tuteja, 2010a,b). The antioxidant protection exerted by exogenous SPM on wheat leaves seemed to be related to the protection of membrane integrity through lipid stabilization and the avoidance of leakage of solutes in Cd2+-induced oxidative damage. On the other hand, Kurepa et al. (1998) reported that paraquat resistance did not necessarily correlate with increased PA content. Lovaas (1997) suggested that the antioxidative effect of PAs is due to a combination of their anion- and cation-binding properties.The binding of PAs to anions (phospholipid membranes, nucleic acids) contributes

Alleviation of Cadmium Stress in Wheat by Polyamines

487

to a high local concentration at particular sites prone to oxidation, whereas the binding to cations efficiently prevents site-specific generation of “active oxygen” (hydroxyl radicals and singlet oxygen).

3.8 Effects of Seed Soaking and Foliar Spraying With Polyamines on the Yield and Components of CadmiumStressed Wheat Plants Data in Fig. 17.3 show that Cd2+-treated wheat plants exhibited significant reductions in yield and its components (i.e., spike length, number of spikelets per spike, number of grains per spike, grains weight per plant, and 1000grain weight) compared with those of control plants over both the 2013–14 and 2014–15 seasons. Under Cd2 stress, SPM-, SPD-, and PUT-pretreated plants showed significant increases in spike length, number of spikelets per spike, number of grains per spike, grain weight per plant, and 1000-grain weight compared with corresponding untreated plants in both growing seasons. Except for the parameter of 1000-grain weight, which correlated with SPD, PUT was the most effective pretreatment, mitigating the injurious effects of Cd2+ stress and significantly increasing yield characteristics compared with Cd2+-stressed plants. The increases were 98.0%, 36.9%, 17.4%, 79.0%, and 48.4%, respectively for the first season under Cd stress compared with controls, and were 92.9%, 29.1%, 28.0%, 91.9%, and 72.3%, respectively for the second season under Cd stress compared with controls. Cd2+-treated wheat plants exhibited significant reductions in their yield and its components. These reductions can be attributed to the decrease in total cumulative leaf area, photosynthetic pigments, polysaccharides and nitrogenous compounds in leaves, and consequently in wheat grain yield (Aldesuquy et al., 2011). These results agree well with those obtained by Mallan and Farrant (1998). Decreases in yield and yield components in different crops under similar conditions have also been reported by many workers (Mallan and Farrant, 1998; Aldesuquy et al., 2004). It has been shown in the present study that exogenous PA applications alleviated the adverse effects of Cd2+ stress on the yield and yield components of wheat plants, leading to increases in both parameters. These increases in yield production by PAs may be due to the increased longevity of leaves, which perhaps contributed to grain filling by enhancing the duration of photosynthates supply to grains (Kaur-Sawhney et al., 1982).This phenomenon was manifested particularly when it was found that there was a positive correlation between phloem area in both the flag leaf and peduncle of the main shoot of wheat plants, which accelerates rapid translocation of photoassimilates from source (i.e.

488

Cadmium Tolerance in Plants: Agronomic, Molecular, Signaling, and Omic Approaches

Figure 17.3  Effect of polyamines on grain yield and its components of Cd-stressed wheat plants. *Control means plants without treatment, neither Cd nor PAs. LSD = spike length, 0.52 and 0.70; number of spikelets per spike, 0.37 and 0.38; number of grains per spike, 0.27 and 0.27; 1000-grain weight, 3.50 and 3.50; and grain weight per plant, 0.05 and 0.06; for two seasons, respectively.

Alleviation of Cadmium Stress in Wheat by Polyamines

489

Figure 17.4  Effect of polyamines on grain Cd content and WUE of Cd-stressed wheat plants. *Control means plants without treatment, neither Cd nor PAs. LSD = grain Cd, 0.13 and 0.15; and WUE, 0.51 and 0.50; for two seasons, respectively.

flag leaf) to sink (i.e. grain in spike) (Aldesuquy, 2014). In this respect, PAs play very important roles in many physiological processes (related to yield quality) such as reproductive organ development, tuberization, floral initiation, and fruit development and ripening (Tiburcio et al., 2002).

3.9 Effects of Seed Soaking and Foliar Spraying With Polyamines on Grain Cadmium Content and Water Use Efficiency in Cadmium-Stressed Wheat Plants Data in Fig. 17.4 show that Cd2+-treated plants exhibited significant increases in the Cd content of grains compared with control over both the 2013–14 and 2014–15 seasons.The increase in Cd content was 99.1% in the

490

Cadmium Tolerance in Plants: Agronomic, Molecular, Signaling, and Omic Approaches

first season and 98.4% in the second under Cd stress compared with control. Under Cd2+ stress, SPM-, SPD-, and PUT-treated plants showed significant decreases in Cd content in grains. SPD, used for seed soaking, was the most efficient pretreatment, mitigating the injurious effects of Cd2 stress and significantly decreasing Cd content in grains compared with Cd2-stressed plants that were not applied with PAs. WUE is the ability of a crop to produce biomass per unit of water transpired (Liang et al., 2006) or the efficiency for producing dry matter per unit of absorbed water and the ability to allocate an increased proportion of biomass into grains (Manivannan et al., 2007).Water scarcity stress is a major limiting factor in agricultural production worldwide (Liu et al., 2005; Shao et al., 2008).Values of WUEG in Cd2+-treated plants were significantly lower than those of the control. This decreased WUEG is probably due to the decreased grain yield of wheat plants (Fig. 17.4). Under Cd2+ stress, SPM, SPD, and PUT treatment mitigated the harmful effects of wastewater stress on the WUEG of wheat plants. The improvement of WUE in nonstressed and stressed wheat plants under PA treatment might be due to increases in the grain yields of wheat plants. Furthermore, the increase in WUEG values was higher with PUT treatment, which was the most effective pretreatment, mitigating the injurious effects of Cd2+ stress and with significantly higher increased yield characteristics than those of others. Yield is a result of the integration of metabolic reactions in plants; consequently, any factor that influences this metabolic activity at any period of plant growth can affect crop yield (Ibrahim and Aldesuquy, 2003).

4. CONCLUSIONS Exogenous applications of SPM, SPD, and PUT have been shown to improve the activities of some key antioxidative enzymes and the contents of nonenzymatic antioxidants, as well as the contents of osmoprotectants and mineral nutrients in wheat seedlings grown under Cd stress. However, the effects of exogenous SPD and PUT for stimulating the plant antioxidative defense systems are greater under Cd stress, suggesting that this improved antioxidant activity may be responsible, at least in part, for the greater tolerance of SPD- and PUT-treated wheat seedlings under Cd stress, thus protecting the antioxidative defense system and leading to improved wheat plant growth and productivity as well as yield quality.

Alleviation of Cadmium Stress in Wheat by Polyamines

491

REFERENCES Ahmad, P., Nabi, G., Ashraf, M., 2011. Cadmium-induced oxidative damage in mustard [Brassica juncea (L.) Czern. and Coss.] plants can be alleviated by salicylic acid. S. Afr. J. Bot. 77, 36–44. Aldesuquy, H.S., 2014. Effect of spermine and spermidine on wheat plants irrigated with waste water: conductive canals of flag leaf and peduncle in relation to grain yield. J. Stress Physiol. Biochem. 10, 145–166. Aldesuquy, H.S., Haroun, S.A., Abo-Hamed, S.A., Al-Saied, A.A., 2011. Physiological studies of some polyamines on wheat plants irrigated with waste water. Osmolytes in relation to osmotic adjustment and grain yield. Phyton 50, 263–268. Aldesuquy, H.S., Haroun, S.A., Abo-Hamed, S.A., El-Said, A.A., 2004. Ameliorating effect of kinetin on pigments, photosynthetic characteristics, carbohydrate contents and productivity of cadmium treated Sorghum bicolor plants. Acta Bot. Hung. 46, 1–21. Alscher, P.G., Erturk, N., Heath, L.S., 2002. Role of superoxide dismutases (SODs) in controlling oxidative stress in plant. J. Exp. Bot. 53, 1331–1341. Alsokari, S.S., Aldesuquy, H.S., 2011. Synergistic effect of polyamines and waste water on leaf turgidity, heavy metals accumulation in relation to grain yield. J. Appl. Sci. Res. 7 (3), 376–384. Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism oxidatives and signal transduction. Ann. Rev. Plant Physiol. Plant Mol. Biol. 55, 373–399. Aravind, P., Prasad, M.N.V., 2005. Modulation of cadmium-induced oxidative stress in Ceratophyllum demersum by zinc involves ascorbate–glutathione cycle and glutathione metabolism. Plant Physiol. biochem. 43, 107–116. Baryla, A.P., Carrier, F., Franck, C., Coulomb, C., Sahut, M., 2001. Havaux, leaf chlorosis in oilseed rape plants (Brassica napus) grown on cadmium-polluted soil: causes and consequences for photosynthesis and growth. Planta 212, 696–709. Bashri, G., Prasad, S.M., 2015. Indole acetic acid modulates changes in growth, chlorophyll a fluorescence and antioxidant potential of Trigonella foenum-graecum L. grown under cadmium stress. Acta Physiol. Plant 37, 1745. Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for water stress studies. Plant Soil 39, 205–207. Baumann, H.A., Morrison, L., Stengel, D.B., 2009. Metal accumulation and toxicity measured by PAM Chlorophyll fluorescence in seven species of marine macroalgae. Ecotoxicol. Environ. Saf. 72, 1063–1075. Beer, R.F., Sizer, I.W., 1952. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195, 133. Besford, R.T., Richardson, C.M., Campos, J.L., Tiburcio, A.F., 1993. Effect of polyamines on stabilization of molecular complexes of thylakoid membranes of osmotically stressed oat leaves. Planta 189, 201–206. Borell, A., Carbonell, L., Farras, R., Puig-Parellada, P.,Tiburcio, A.F., 1997. Polyamines inhibit lipid peroxidation in senescing oat leaves. Physiol. Plant 99, 385–390. Bouchereau, A., Aziz, A., Larher, F., Martin-Tanguy, J., 1999. Polyamines and environmental challenges: recent development. Plant Sci. 140, 103–125. Boussama, N., Quariti, O., Ghorbal, M.H., 1999. Changes in growth and nitrogen assimilation in barley seedlings under cadmium stress. J. Plant Nutr. 22, 731–752. Cakmak, I., Marshner, H., 1992. Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase and glutathione reductase in bean leaves. Plant Physiol. 98, 1222–1227. Chmielowska-Bąk, J., Lefèvre, I., Lutts, S., Deckert, J., 2013. Short term signaling responses in roots of young soybean seedlings exposed to cadmium stress. J. Plant Physiol. 170, 1585–1594.

492

Cadmium Tolerance in Plants: Agronomic, Molecular, Signaling, and Omic Approaches

Cho, M., Chardonnens, A.N., Dietz, K.J., 2003. Differential heavy metal tolerance of Arabidopsis halleri and Arabidopsis thaliana: a leaf slice test. New Phytol. 158, 287–293. Choudhury, S., Panda, S.K., 2004. Role of salicylic in regulating cadmium induced oxidative stress in Oryza sativa roots. Bulg. J. Plant Physiol. 30, 95–110. Clemens, S., Antosiewicz, D.M.,Ward, J.M., Schachtman, D.P., Schroeder, J.I., 1998.The plant cDNA LCT1 mediates the uptake of calcium and cadmium in yeast. Proc. Nat. Acad. Sci. U.S.A. 95, 12043–12048. Cohen, S.S., 1998. A Guide to the Polyamines. Oxford University Press, New York, NY. Das, P., Samantaray, S., Rout, G.R., 1997. Studies on cadmium toxicity in plants: a review. Environ. Pollut. 98, 29–36. De Maria, S., Puschenreiter, M., Rivelli, A.R., 2013. Cadmium accumulation and physiological response of sunflower plants to Cd during the vegetative growing cycle. Plant Soil Environ. 59, 254–261. Deng, G., Li, M., Li, H.,Yin, L., Li,W., 2014. Exposure to cadmium causes declines in growth and photosynthesis in the endangered aquatic fern (Ceratopteris pteridoides). Aquat. Bot. 112, 23–32. Dewdar, M.D., El-Yazal, M.A., El-Ganaini, S.S., 2008. Effect of biofertilization and optimization of nitrogen fertilizer on vegetative growth, chemical composition, yield and yield components trails of wheat plants. Egypt. J. Appl. Sci. 23 (4B), 486–501. Drolet, G., Dumbroff, E.B., Legg, R.,Thompson, J.E., 1986. Radical scavenging properties of polyamines. Phytochemistry 25, 367–371. El-Bassiouny, H.M.S., Mostafa, H.A., El-Khawas, S.A., Hassanein, R.A., Khalil, S.I., Abd ElMonem, A.A., 2008. Physiological responses of wheat plant to foliar treatments with arginine or putrescine. Austr. J. Basic Appl. Sci. 2 (4), 1390–1403. Eller, M.H., Warner, A.L., Knap, H.T., 2006. Genomic organization and expression analyses of putrescine pathway genes in soybean. Plant Physiol. Biochem. 44, 49–57. Fecenko, J., Lozek, o., filipek-mazur, B., Mazur, K., 1997. Cadmium uptake and localization in spring barley after application of sodium humate. Zesz. Probl. Post. Nauk. Roln. 448a, 83. Gill, S.S., Tuteja, N., 2010a. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol. Biochem. 48, 909–930. Gill, S.S., Tuteja, N., 2010b. Polyamines and abiotic stress tolerance in plants. Plant Signal. Behav. 5, 26–33. Gonçalves, J.F., Antes, F.G., Maldaner, J., Pereira, L.B.,Tabaldi, L.A., Rauber, R., Rossato, L.V., Bisognin, D.A., Dressler, V.L., Flores, E.M., Nicoloso, F.T., 2009. Cadmium and mineral nutrient accumulation in potato plants grown under cadmium stress in two different experimental culture conditions. Plant Physiol. Biochem. 47 (9), 814–821. Groppa, M.D., Benavides, M.P., 2008. Polyamines and abiotic stress: recent advances. Amino Acids 1, 35–45. Groppa, M.D., Benavides, M.P.,Tomaro, M.L., 2003. Polyamine metabolism in sunflower and wheat leaf discs under cadmium or copper stress. Plant Sci. 161, 481–488. Groppa, M.D., María, L.T., Benavides, M.P., 2001. Polyamines as protectors against cadmium or copper-induced oxidative damage in sunflower leaf discs. Plant Sci. 161, 481–488. Groppa, M.D., Tomaro, M.L., Benavides, M.P., 2007. Polyamines and heavy metal stress: the antioxidant behavior of spermine in cadmium- and copper-treated wheat leaves. Biometals 20, 185–195. Gussarson, M., Jensen, P., 1992. Effects of copper and cadmium on uptake and leakage of K+ in Birch (Betula pendula) Roots. Tree Physiol. 11 (3), 305–313. Hafez, A., Mikkelsen, D.S., 1981. Colorimetric determination of nitrogen for evaluating the nutritional status of rice. Commun. Soil Sci. Plant Anal. 12, 61–69.

Alleviation of Cadmium Stress in Wheat by Polyamines

493

Harpaz-Saad, S.,Azoulay,T.,Arazi,T., Ben-Yaakov, E., Mett,A., Shiboleth,Y.M., Hortensteiner, S., Gidoni, D., Gal-On, A., Goldschmidt, E.E., Eyal, Y., 2007. Chlorophyllase is a ratelimiting enzyme in chlorophyll catabolism and is posttranslationally regulated. Plant Cell 19 (3), 1007–1022. Hernandez-Lopez, J., Gollas-Galvan,T.,Vargas-Albores, F., 1996. Activation of the prophenoloxidase system of the brown shrimp Ž .Penaeus californiensis Holmes. Comp. Biochem. Physiol. 113C, 61–66. Hoagland, D.R., Arnon, D.I., 1950. The water-culture for growing plants without soil. Cal. Agric. Exp. Sta Cir. 347, 1–32. Hsu,Y.T., Kao, C.H., 2007. Cadmium-induced oxidative damage in rice leaves is reduced by polyamines. Plant Soil 291, 27–37. Ibrahim, A.H., Aldesuquy, H.S., 2003. Glycine betaine and shikimic acid induced modification in growth criteria, water relation and productivity of drought Sorghum bicolor plants. Phyton 43, 351–363. Jackson, M.L., 1967. Soil Chemical Analysis. Prentice Hall of India Private limited, New Delhi, India. Jalil, A., Selles, F., Clarke, J.M., 1994. Effect of cadmium on growth and the uptake of cadmium and other elements by durum wheat. Plant Nutr 17, 1839–1858. Jha, A.B., Dubey, R.S., 2004. Carbohydrate metabolism in growing rice seedlings under arsenic toxicity. Plant Physiol. 123, 1029–1036. Kabata-Pendias, A., Pendias, H., 1992. Trace Elements in Soils and Plants, second ed. CRC Press, Boca Ratón, Florida, p. 315. Kaur-Sawhney, R., Shih-Flores, H.E., Galston, A.W., 1982. Relation of polyamine synthesis and titer to aging and senescence in oat leaves. Plant Physiol. 69, 405–410. Khurana, M.P., Jhanji, S., 2014. Influence of cadmium on dry matter yield, micronutrient content and its uptake in some crops. J. Environ. Biol. 35, 865–870. Kopittke, P.M., Pax, F., Blamey, C., Colin, J., Neal, A., Menzies, W., 2010. Trace metal phytotoxicity in solution culture: a review. J. Exp. Bot. 61, 945–954. Kubis, J., Floryszak-Wieczorek, J., Arasimowicz-Jelonek, M., 2014. Polyamines induce adaptive responses in water deficit stressed cucumber roots. J. Plant Res. 127, 151–158. Kumar, M., 2013. Crop plants and abiotic stresses. J. Biomol. Res. Ther. 3 (1), e125. Kumar, M., Bijo, A.J., Baghel, R.S., Reddy, C.R.K., Jha, B., 2012. Selenium and spermine alleviate cadmium induced toxicity in the red seaweed Gracilaria dura by regulating antioxidants and DNA methylation. Plant Physiol. Biochem. 51, 129–138. Kurepa, J., Smalle, J., Vanmontagu, M., Inze, D., 1998. Polyamines and paraquat toxicity in Arabidopsis thaliana. Plant Cell Physiol. 39, 987–992. Kuznetsov,V.L., Shevyakova, N.I., 2010. Polyamines and plant adaptation to saline environments. In: Remawat, K.G. (Ed.), Desert Plants. Springer,Verlag Berlin-Heidelberg, pp. 261–298. Li, C.L., Xu, H.M., Xu, J., Chun, X.Y., Ni, D.J., 2011. Effects of aluminum on ultrastructure and antioxidant activity in leaves of tea plant. Acta Physiol. Plant 33, 973–978. Liang, Z.S., Yang, J.W., Shao, H.B., Han, R.L., 2006. Investigation on water consumption characteristics and water use efficiency of Poplar under soil water-deficits on the Loess Plateau. Biointerfaces 53, 23–28. Lindberg, S., Wingstrand, G., 1985. Mechanisms for Cd2+ inhibition of (K++, Mg2+) ATPase activity and uptake in roots of sugar beet (Beta vulgaris). Physiol. Plant 63, 181–186. Liu, H., Yu, B.J., Zhang, W., Liu, Y., 2005. Effect of osmotic stress on the activity of H+ATPase and the levels of covalently and non-covalently conjugated polyamines in plasma membrane preparation from wheat seedling roots. Plant Sci. 168, 1599–1607. Lovaas, E., 1997. Antioxidant and metal-chelating effects of polyamines. In: Sies, H. (Ed.), Advances in Pharmacology. Antioxidants in Disease Mechanisms and Therapy, 38. Academic Press, pp. 119–149.

494

Cadmium Tolerance in Plants: Agronomic, Molecular, Signaling, and Omic Approaches

Maehly, A.C., Chance, B.C., 1954. The assay of catalases and peroxidases. In: Glick, D. (Ed.), Methods of Biochemical Analysis,Vol. 1. Interscience Publish, New York. Mallan, H.I., Farrant, J.M., 1998. Effect of metal pollutants cadmium and nickel on soybean seed development. Seed Sci. Res. 8, 445–453. Manivannan, P., Jaleel, C.A., Sankar, B., Kishorekumar, A., Somasundaram, R., Lakshmanan, G.M.A., Panneerselvam, R., 2007. Growth biochemical modifications and proline metabolism in Helianthus annuusL. As induced by drought stress. Colloids Surf. B Biointerfaces 59, 141–149. Metwali, M.R., Gowayed, S.M.H., Al-Maghrabi, O.A., Mosleh, Y.Y., 2013. Evaluation of toxic effect of copper and cadmium on growth, physiological traits and protein profile of wheat (Triticum aestivum L.), maize (Zea mays L.) and sorghum (Sorghum bicolor L.). World Appl. Sci. J. 21, 301–314. Minocha, R., Majumdar, R., Minocha, S.C., 2014. Polyamines and abiotic stress in plants: a complex relationship. Front. Plant Sci. 5, 175. Mohan, B.S., Hosetti, B.B., 1999. Aquatic plants for toxicity assessment. Environ. Res. Sect. A 81, 259–274. Nahar, K., Hasanuzzaman, M., Alam, M.M., Rahman, A., Suzuki, T., Fujita, M., 2016. Polyamine and nitric oxide crosstalk: antagonistic effects on cadmium toxicity in mung bean plants through upregulating the metal detoxification, antioxidant defense and methylglyoxal detoxification systems. Ecotoxicol. Environ. Saf. 126, 245–255. Nazar, R., Iqbal, N., Masood, A., Khan, M.I.R., Syeed, S., Khan, N.A., 2012. Cadmium toxicity in plants and role of mineral nutrients in its alleviation. Am. J. Plant Sci. 3, 1476–1489. Osman, A.S., Rady, M.M., 2014. Effect of humic acid as an additive to growing media to enhance the production of eggplant and tomato transplants. J. Hortic. Sci. Biotechnol. 89 (3), 237–244. Ouyang, H., Kong, X., He, W., Qin, N., He, Q., Wang, Y., Wang, R., Xu, F., 2012. Effects of five heavy metals at sub-lethal concentrations on the growth and photosynthesis of Chlorella vulgaris. Chin. Sci. Bull. 57, 3363–3370. Ouzounidou, G., Moustakas, M., Eleftheriou, E.P., 1997. Physiological and ultrastructural effects of cadmium on wheat (Triticum aestivum L.) leaves. Arch. Environ. Contam. Toxicol. 32, 154–160. Page, A.I., Miller, R.H., Keeney, D.R., 1982. Methods of Soil Analysis. Part 2: Chemical and Microbiological Properties, second ed. Amer. Soc. Agron., Madison, Wisconsin, USA. Pandey, P., Irulappan, V., Bagavathiannan, M.V., Kumar, M.S., 2017. Impact of combined abiotic and biotic stresses on plant growth and avenues for crop improvement by exploiting physio-morphological traits. Front. Plant Sci. 8, 537. Pena, L.B., Tomaro, M.L., Gallego, S.M., 2006. Effect of different metals on protease activity in sunflower cotyledons. Electron. J. Biotechnol. 9, 3–18. Piper, C.S., 1947. Soil and Plant Analysis. Interscience Publishers, Inc., New York, NC, USA. Poduslo, J.F., Curran, G.L., 1996. Increased permeability of superoxide dismutase at the blood-nerve and blood–brain barriers with retained enzymatic activity after modification with naturally occurring polyamine, putrescine. J. Neurochem. 67, 734–741. Premchandra, G.S., Saneoka, H., Ogata, S., 1990. Cell membrane stability, an indicator of drought tolerance as affected by applied nitrogen in soybean. J. Agric. Sci. Camb. 115, 63–66. Quartacci, M.F., Cosi, E., Navari-Izzo, F., 2001. Lipids and NADPH-dependent superoxide production in plasma membrane vesicles from roots of wheat grown under copper deficiency or excess. J. Exp. Bot. 52, 77–84. Rady, M.M., 2011. Effect of 24-epibrassinolide on growth, yield, antioxidant system and cadmium content of bean (Phaseolus vulgaris L.) plants under salinity and cadmium stress. Sci. Hortic. 129, 232–237.

Alleviation of Cadmium Stress in Wheat by Polyamines

495

Rady, M.M., Hemida, K.A., 2015. Modulation of cadmium toxicity and enhancing cadmium-tolerance in wheat seedlings by exogenous application of polyamines. Ecotoxicol. Environ. Saf. 119, 178–185. Rady, M.M., Seif El-Yazal, M.A., Taie, H.A.A., Abdel-Mageed, S.M.A., 2016. Response of Triticum aestivum (L.) plants grown under cadmium stress to polyamines pretreatments. Plant 4 (5), 29–36. Rascio, N.,Vecchia, F.D., Ferretti, M., Merlo, L., Ghisi, R., 1993. Some effects of cadmium on maize plants. Environ. Contam. Toxicol. 25, 244–249. Rhee, H.J., Kim, E.J., Lee, J.K., 2007. Physiological polyamines: simple primordial stress molecules. J. Cell Mol. Med. 11, 685–703. Rider, J.E., Hacker, A., Mackintosh, C.A., Pegg, A.E., Woster, P.M., Casero Jr., R.A., 2007. Spermine and spermidine mediate protection against oxidative damage caused by hydrogen peroxide. Amino Acids 33, 231–240. Schutzendubel, A., Polle, A., 2002. Plant responses to abiotic stresses: heavy metal induced oxidative stress and protection by mycorrhization. J. Exp. Bot. 53, 1351–1365. Scott,T.A., Melvin, E.H., 1956. Anthrone colorimetric method. In:Whistler, R.L.,Walfrom, M.L. (Eds.), Methods in Carbohydrate Chemistry, 1. Academic Press, New York, London, p. 384. Semida,W.M., Rady, M.M., Abd El-Mageed,T.A., Howladar, S.M., Abdelhamid, M.T., 2015. Alleviation of cadmium toxicity in common bean (Phaseolus vulgaris L.) plants by the exogenous application of salicylic acid. J. Hortic. Sci. Biotechnol. 90, 83–91. Shao, H.B., Chu, L.Y., Jaleel, A.C., Zhao, C.X., 2008.Water-deficit stress-induced anatomical changes in higher plants. C. R. Biol. 331, 215–225. Sharma, P., Dubey, R.S., 2005. Lead toxicity in plants. Braz. J. Plant Physiol. 17, 35–52. Sharma, S.S., Dietz, K.J., 2006.The significance of amino acids and amino acid-derived molecules in plant responses and adaptation to heavy metal stress. J. Exp. Bot. 57, 711–726. Sharmila, P., Pardha Saradhi, P., 2002. Proline accumulation in heavy metal stressed plants: an adaptive strategy. In: Prasad, M.N.V., Strzalka, K. (Eds.), Physiology and Biochemistry of Metal Toxicity and Tolerance in Plants, pp. 179–199. Shevyakova, N.I., Il’ina, E.N., Stetsenko, L.A., Kuznetsov, V.I.V., 2010. Nickel accumulation in rape shoots (Brassica Napus L.) increased by putrescine. Int. J. Phytoremed. 13 (4), 345–356. Stanhill, G., 1987. Water use efficiency. Adv. Agron. 39, 53–85. Sullivan, C.Y., Ross,W.M., 1979. Selecting the drought and heat resistance in grain sorghum. In: Mussel, H., Staples, R.C. (Eds.), Stress Physiology in Crop Plants. John Wiley & Sons, New York, pp. 263–281. Szepesi, A., 2006. Salicylic acid improves the acclimation of Lycopersicon esculentum Mill. L. to high salinity by approximating its salt stress response to that of the wild species L. pennellii. Acta Biol. Szeged. 50 (3–4), 177. Tang, C.F., Liu,Y.G., Zeng, G.M., Li, X., et al., 2005. Effects of exogenous spermidine on antioxidant system responses of Typha latifolia L. under Cd2+ stress. J. Int. Plant Biol. 47, 428–434. Tiburcio, A.F., Altabella, T., Masgrau, C., 2002. Polyamines. In: Bisseling, T., Schell, J. (Eds.), New Developments in Plant Hormone Research. Springer-Verlag, New York. Tiburcio, A.F., Kaur-Sawhney, R., Galston, A.W., 1990. Polyamine metabolism. In: Miflin, B.J., Lea, P.J. (Eds.), Intermedatory Nitrogen Metabolism. 16, the Biochemistry of Plants. Academic Press, pp. 283–325. Tlustos, P., Balik, J., Pavlikova, D., Szakova, J., 1997.The uptake of cadmium, zinc, arsenic and lead by chosen crops. Rostl.Vyroba. 43 (10), 487. Van Assche, F., Clijsters, H., 1990. Effects of metals on enzyme activity in plants. Plant Cell Environ. 13, 195–206. Vantová, I., Bačkor, M., Klejdus, B., Bačkorová, M., Kováčik, J., 2013. Copper uptake and copper induced physiological changes in the epiphytic lichen Evernia prunastri. Plant Growth Regul. 69, 1–9. Vassilev, A.,Yordanov, I.,Tsonev,T., 1997. Effects of Cd2+ on the physiological state and photosynthetic activity of young barely plants. Photosynthesis 34, 293–302.

496

Cadmium Tolerance in Plants: Agronomic, Molecular, Signaling, and Omic Approaches

Velikova, V., Yordanov, I., Edreva, A., 2000. Oxidative stress and some antioxidant system in acid rain treated bean plants: protective role of exogenous polyamines. Plant Sci. 151, 59–66. Veselov, D., Kudoyarova, G., Symonyan, M., Veselov, S., 2003. Effect of cadmium on ion uptake, transpiration and cytokinin content in wheat seedlings. Bulg. J. Plant Physiol. (Special Issue), 353–359. Walden, R., Alexandra, C.,Tiburcio, A.F., 1997. Polyamines: small molecules triggering pathways in plant growth and development. Plant Physiol. 113, 1009–1013. Walters, D.R., 2003. Polyamines and plant disease. Phytochemistry 64, 97–107. Wang, M., Zheng, Q., Shen, Q., Guo, S., 2013. The critical role of potassium in plant stress response – a review. Int. J. Mol. Sci. 14, 7370–7390. Weatherly, P.E., 1950. Studies in the water relations of cotton. 1. The field measurement of water deficits in leaves. New Phytol. 49, 81–97. Weinstein, L.H., Kaur-Sawney, R.K., Rajam, M.V., Wettlanfer, S.H., Galston, A.W., 1986. Cadmium-induced accumulation of putrescine in oat and bean leaves. Plant Physiol. 82, 641–645. Welburn, A.R., Lichtenthaler, H., 1984. Formulae and program to determine total carotenoids and chlorophylls a and b leaf extracts in different solvents. In: Sybesma, C. (Ed.), Advances in Photosynthesis Research 2, pp. 9–12. Xiong, Z., Zhao, F., Li, M., 2006. Lead toxicity in Brassica pekinensis Rupr.: effect on nitrate assimilation and growth. Environ. Toxicol. 21 (2), 147–153. Yadav, S.K., 2010. Heavy metals toxicity in plants: an overview on the role of glutathione and phytochelatins in heavy metal stress tolerance of plants. S. Afr. J. Bot. 76, 167–179. Yan, Z., Li, X., Chen, J., Tam, N., 2015. Combined toxicity of cadmium and copper in Avicennia marina seedlings and the regulation of exogenous jasmonic acid. Ecotoxicol. Environ. Saf. 113, 124–132. Yang, J.C., Zhang, J.H., Liu, K.,Wang, Z.Q., Liu, L.J., 2007. Involvement of polyamines in the drought resistance of rice. J. Exp. Bot. 58, 1545–1555. Yildiz, N., 2005. Response of tomato and corn plants to increasing Cd levels in nutrient culture. Pak. J. Bot. 37 (3), 593–599. Yiu, J.-C., Juang, L.-D., Fang, D.Y.-T., Liu, C.-W., Wu, S.-J., 2009. Exogenous putrescine reduces flooding-induced oxidative damage by increasing the antioxidant properties of Welsh onion. Sci. Hortic. 120, 306–314. Zaki, E., Nabila, M., Hassanein, M.S., Gamal El-Din, K., 2007. Growth and yield of some wheat cultivars irrigated with saline water in newly cultivated land as affected by biofertilization. J. Appl. Sci. Res. 3 (10), 1121–1126. Zhang,Y., Xu, S.,Yang, S., Chen,Y., 2015. Salicylic acid alleviates cadmium-induced inhibition of growth and photosynthesis through upregulating antioxidant defense system in two melon cultivars (Cucumis melo L.). Protoplasma 252, 911–924. Zhao, H.,Yang, H., 2008. Exogenous polyamines alleviate the lipid peroxidation induced by cadmium chloride stress in Malus hupehensis Rehd. Sci. Hortic. 116, 442–447. Zheng, G., Lv, H.P., Gao, S., Wang, S.R., 2010. Effects of cadmium on growth and antioxidant responses in Glycyrrhiza uralensis seedlings. Plant Soil Environ. 56, 508–515. Zornoza, P.,Vázquez, S., Esteban, E., Fernández-Pascual, M., Carpena, R., 2002. Cadmiumstress in nodulated white lupin: strategies to avoid toxicity. Plant Physiol. Biochem. 40 (12), 1003–1009.

FURTHER READING Ferreira, R.R., Fornazeir, R.F.,Vitoria, A.P., Lea, A.P., Azevedo, R.A., 2002. Changes in antioxidant enzymes activities in soybean under cadmium stress. Plant Nutr. 25, 327–342.