Research Article Received: 17 January 2014
Accepted article published: 2 April 2014
Published online in Wiley Online Library: 15 May 2014
(wileyonlinelibrary.com) DOI 10.1002/ps.3789
Thiamethoxam as a seed treatment alters the physiological response of maize (Zea mays) seedlings to neighbouring weeds Maha Afifi, Elizabeth Lee, Lewis Lukens and Clarence Swanton* Abstract BACKGROUND: Thiamethoxam is a broad-spectrum neonicotinoid insecticide that, when applied to seed, has been observed to enhance seedling vigour under environmental stress conditions. Stress created by the presence of neighbouring weeds is known to trigger the accumulation of hydrogen peroxide (H2 O2 ) in maize seedling tissue. No previous work has explored the effect of thiamethoxam as a seed treatment on the physiological response of maize seedlings emerging in the presence of neighbouring weeds. RESULTS: Thiamethoxam was found to enhance seedling vigour and to overcome the expression of typical shade avoidance characteristics in the presence of neighbouring weeds. These results were attributed to maintenance of the total phenolics content, 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activity and anthocyanin and lignin contents. These findings were also associated with the activation of scavenging genes, which reduced the accumulation of H2 O2 and the subsequent damage caused by lipid peroxidation in maize seedlings originating from treated seeds even when exposed to neighbouring weeds. CONCLUSIONS: These results suggest the possibility of exploring new chemistries and modes of action as novel seed treatments to upregulate free radical scavenging genes and to maintain the antioxidant system within plants. Such an approach may provide an opportunity to enhance crop competitiveness with weeds. © 2014 Society of Chemical Industry Keywords: R:FR; corn; morphology; DPPH radical scavenging; anthocyanin; lignin; hydrogen peroxide; free radicals; lipid peroxidation; scavenging genes; antioxidant system
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INTRODUCTION
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Thiamethoxam has been reported to increase the phenolics content and to induce the antioxidant capacity of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging in maize.2 Thiamethoxam also altered expression of genes involved in plant defences and regulated by jasmonic acid (JA), salicylic acid (SA) or both pathways against spider mites in cotton, corn and tomato.8 In Arabidopsis, Ford et al.9 reported that thiamethoxam elevated the level of SA within treated plants. SA is an antioxidant that plays an important role in the defence against plant pathogens and is also known to modulate the response of plants to abiotic and biotic stresses. Bi et al.10 tested thiamethoxam for the control of whitefly, a serious pest of strawberry. They found that thiamethoxam maintained soluble solids and glucose levels within the fruit. They also reported that thiamethoxam increased ascorbic acid within the fruit, thereby enhancing antioxidant activity. Increasing the antioxidant activity would account for the observed enhancement in seedling vigour.11 Plant cells have
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Correspondence to: Clarence Swanton, Department of Plant Agriculture, Crop Science Building, University of Guelph, 50 Stone Road E., Guelph, ON N1G 2 W1, Canada. E-mail: [email protected] Department of Plant Agriculture, University of Guelph, Guelph, ON, Canada
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Thiamethoxam [3-(2-chloro-1,3-thiazol-5-ylmethyl)-5-methyl-1,3, 5-oxadiazinan-4-ylidene(nitro)amine] is a broad-spectrum neonicotinoid insecticide.1 This compound controls a wide variety of commercially important crop pests with low use rates and flexible application methods.2 It is commercially used either as a foliar spray or as a soil treatment under the trademark Actara™ and as a seed treatment under the trademark Cruiser®.1,3 When this chemistry is applied as a seed treatment, thiamethoxam is distributed systematically throughout the plant, the concentration decreasing with increasing biomass.4 The use of thiamethoxam as a seed treatment reduces human exposure and the potential for off-target movement of this chemistry into the environment.3 Thiamethoxam applied to seed has been observed to increase seed germination, root growth, seedling height and biomass accumulation.2,5 For example, Cataneo et al.6 reported that thiamethoxam promoted seed germination and suggested that this may reduce the time for crop establishment. The use of thiamethoxam was also found to increase root development and to alter the distribution of photoassimilates.5,7 The physiological mechanism by which this germination and growth enhancement occur is not well known. Such physiological changes may be the result of modification of the metabolic pathways within the seedling.
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evolved antioxidative systems that enable the removal of free radicals.12 – 14 This system includes enzymes such as superoxide dismutase, (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and glutathione S-transferase, (GST). In addition, non-enzymatic compounds such as ascorbic acid, phenols and flavonoids such as anthocyanins are known to be involved in antioxidant defence systems. Polyphenols such as flavonoids can chelate transition metal ions, directly scavenge reactive oxygen species (ROS), delay diffusion of free radicals, limit peroxidative reactions and inhibit lipid peroxidation.15 For example, transgenic potato plants with an increased concentration of flavonoid showed improved antioxidant capacity.16 These phenolic compounds produced via the phenylpropanoid pathway perform important functions involved in protecting plant development during periods of environmental stress.17,18 Flavonoids such as anthocyanins protect plants against UV irradiation, while salicylic acid is implicated in plant–pathogen interactions.19,20 Anthocyanins are produced in the phenylpropanoid pathway, in which chalcone synthase (CHS) catalyses the formation of the flavonoids from p-coumaroyl-CoA, which leads to the synthesis of anthocyanin. Within this pathway there are two additional subpathways, both of which are initiated from p-coumaroyl-CoA. In both pathways, cinnamyl alcohol dehydrogenase (CAD) is responsible for the production of lignin.21 Lignin is embedded in the plant cell walls and plays an important role in the mechanical support and transport of water in plants. The balance between lignin and anthocyanin produced by this pathway is influenced by environmental conditions.22 – 24 For example, nitrogen limitation can cause a metabolic flux to occur within the phenylpropanoid pathway, resulting in a diversion of anthocyanin production to lignin in the nla Arabidopsis mutant.23 This diversion of anthocyanins to lignin production will reduce the ability of the crop plant to scavenge for free radicals and protect itself against photooxidative damage.12 Oxidative damage caused by ROS includes the alteration of DNA, and the oxidation of proteins and lipids.25 Hydrogen peroxide (H2 O2 ) is a major ROS component, known as an intercellular systemic signalling mechanism, which can alter plant metabolism but is also capable of inflicting cellular damage.14 Accumulation of H2 O2 increases the possibility of the formation of hydroxyl radicals, which may account for subsequent lipid peroxidation. Levels of lipid peroxidation have been used widely as an indicator of ROS-mediated damage to cell membranes under stressful conditions.26,27 During stressful periods, the avoidance of H2 O2 accumulation is an important plant strategy to enhance metabolic recovery. Plants have several mechanisms that can reduce H2 O2 production during a stressful period. These mechanisms include anatomical adaptations and physiological and molecular changes.13 Plants with the ability to scavenge and/or control the level of cellular H2 O2 will be better adapted to survive.14 Efficient scavenging of ROS requires the action of both non-enzymatic and enzymatic scavenging genes.16 An increase in the transcript level of ZmGST1, ZmSOD2, ZmAPX2 and ZmCAT3 genes and modulation of the phenylpropanoid pathway, resulting in a reduction in anthocyanin content, was observed when maize seedlings were exposed to conditions of low R:FR reflected from above-ground neighbouring weeds.24 Accumulation of H2 O2 and a reduction in antioxidant compounds would invariably reduce seedling growth. In contrast, the available literature suggests that seedling vigour was enhanced when seeds were
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treated with thiamethoxam. No previous work has explored the effect of thiamethoxam as a seed treatment on the physiological response of maize seedlings emerging in the presence of neighbouring weeds. Thus, it was hypothesised that this enhancement in growth may be attributed to a reduction in H2 O2 accumulation in seedlings emerging from seed treated with thiamethoxam. It was further hypothesised that thiamethoxam may elevate the expression of genes involved in the scavenging of H2 O2 and the phenylproponoid pathway.
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MATERIALS AND METHODS
2.1 Plant material and growth conditions Maize seeds (Zea mays) of the hybrid NK-N29T-GT (Syngenta Crop Protection Canada Inc.) were selected for study in this experiment. Thiamethoxam-treated seed (100 g AI 100 kg−1 ) and untreated seeds were planted to a depth of 2 cm, one seed per cup, in 8 cm diameter, 10 cm tall, 355 mL plastic cups (Dart Container Corporation, Mason, MI). These cups were filled with Turface, a 100% backed calcined clay growth medium (Turface MVP®; Profile Products LLC, Buffalo Grove, IL). These cups were then placed in 8 cm diameter, 18 cm tall pots (1 L of natural cylinder modified to 18 cm; Consolidated Bottle Co., Toronto, ON). These cylindrical pots were further centred within 25 cm diameter, 19 cm tall, 6 L pots (Airlite Plastics Company, Omaha, NE), as described in Afifi and Swanton.28 The area surrounding the cylindrical pot was filled with Turface, herein referred to as the weed-free treatment. The weedy treatment was seeded into this area of Turface with Fiesta III (Lolium perenne L.), 2–3 weeks prior to planting. This perennial ryegrass was used as a model weed species. Ryegrass was watered as required with Hoagland’s nutrient solution and allowed to grow and form a dense canopy. This canopy was trimmed once to a height of 5 cm prior to the initiation of each replicate to ensure that no shading of the maize seedling occurred. This design ensured that there was no physical root contact between either species or contamination from either root exudates or direct competition for incoming light, water or nutrients. Pots were placed within the same growth cabinet (Converon, Winnipeg, Canada) set to 28/20 ∘ C, 16:8 h light:dark regime and 60–65% humidity. Irradiance was supplied by a sliding bank of Sylvania Cool White fluorescent tubes and inside-frost tungsten 40 W bulbs delivering 500 μmol m−2 s−1 of PPFD. Maize seedlings within each pot were also fertigated daily using Hoagland’s solution. Ten seedlings per treatment (replicated 5 times, N = 50) were harvested at the fourth-leaf-tip stage. Maize seedling height to the second-leaf collar (i.e. the distance from the Turface to the youngest visible collar) and stem diameter 2 cm above the first stem node were measured. Roots were then washed under running tap water and separated from the above-ground stem by cutting just above the highest crown root. The number and length of crown roots for each seedling were recorded. Shoots and the entire root system were then bagged separately and dried at 80 ∘ C to constant weight to determine total shoot and root biomass. For the physiological and molecular analysis, an additional ten seedlings per treatment, replicated 5 times (N = 50), were separated into component parts of the first leaf, stem and crown roots. The remaining three leaves were discarded. Only the first leaf was selected for analysis, based on the results from a previous study.24 These samples were then frozen immediately in liquid nitrogen and stored at −80 ∘ C for further analysis.
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Thiamethoxam alters maize seedlings response to weeds 2.2 Analysis of the total phenolics content in the seedling extracts The total phenolics content in the tissue was determined according to the method described by Singleton et al.29 Briefly, 0.1 g of frozen ground tissue from each seedling component was extracted with 1 mL of absolute methanol. The methanolic mixture was then centrifuged at 12 000 × g for 15 min. An aliquot of (500 μL) of the methanolic extract was mixed with 2.5 mL of 10% Folin–Ciocalteu’s-reagent-dissolved 7.5% NaHCO3 . The mixture was then incubated at 45 ∘ C for 30 min. The absorbance of the mixture was measured at 765 nm against a blank of Folin–Ciocalteu’s reagent that had been prepared similarly but without plant tissue. A calibration curve was prepared using a standard solution of gallic acid, and the results were expressed as μg gallic acid equivalent g−1 fresh weight of sample. 2.3 Analysis of anthocyanin content Total anthocyanin was determined in the tissues according to the method described by Neff and Chory.30 Briefly, 0.1 g of frozen ground tissue from each seedling tissue was extracted with 1 mL of methanol and acidified with 0.1% HCl overnight at 4 ∘ C. After centrifuging the supernatant for 5 min at 5000 × g, anthocyanin was separated from chlorophyll by adding 250 mL of chloroform. The total anthocyanin content was determined by subtracting the absorption values (A) measured at 657 nm from the A values measured at 530 nm of the aqueous phase using a spectrophotometer. 2.4 Analysis of lignin content Lignin was extracted and measured from the ground tissues by the method of Bruce and West.31 A quantity of 3 g of each plant tissue was homogenised separately in 10 mL of 99.5% ethanol, and the extract was centrifuged at 10 000 × g for 15 min. The remaining pellet was freeze dried. A quantity of 50 mg of the dry pellet was extracted and placed in a solution of 5 mL of 2 N HCl and 0.5 mL of thioglycolic acid. This sample was heated at 100 ∘ C for 8 h, cooled and centrifuged again at 14 000 × g for 30 min at 4 ∘ C. The pellet was washed once with 2.5 mL of water and then resuspended in 5 mL of 1 N NaOH. The solution was agitated gently at 25 ∘ C for 18 h. After a further centrifugation at 14 000 × g for 30 min, 1 mL of concentrated HCl was added to the supernatant in a clean test tube and left overnight at 4 ∘ C for lignin thioglycolate to precipitate. Following a final centrifugation at 14 000 × g for 30 min, the pellet was dissolved in 1 mL of 1 N NaOH. The lignin content within the solution was expressed as the A values measured at 280 nm using 1 N NaOH as the blank solution.
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DPPH radical, which was calculated according to the following equation: %Inhibition of DPPH =
Acontrol − Asample Acontrol
× 100
where Acontrol is the absorbance reading of DPPH in the solution without extracts and Asample is the absorbance reading of DPPH within the sample solution. 2.6 Analysis of H2 O2 concentration Hydrogen peroxide in the ground tissues was estimated according to the protocol reported by Patterson et al.33 A quantity of 100 mg of frozen ground tissues was homogenised in 200 μL of cold acetone. After centrifuging for 5 min at 10 000 × g, the supernatant was mixed with 20 μL of titanium reagent (2% TiC12 in concentrated HCl). The Ti–H2 O2 complex was precipitated by adding 40 μL of 15 M ammonia solution. This solution was centrifuged as described above, after which the pellet was washed with cold acetone 2 times and then dissolved in 1 mL of 4 N H2 SO4 . The absorbance of the solution was measured at 410 nm against blanks that had been prepared similarly but without plant tissue. 2.7 Detection of H2 O2 using the DAB staining method Leaves of maize seedlings were cut carefully and placed in plastic tubes containing 1 mg mL−1 of 3,3-diaminobenzidine (DAB)-HCl (Sigma-Aldrich, St Louis, MO). Samples were incubated in the DAB solution overnight. Leaf tissues were placed in boiling ethanol (96%) in order to clear the chlorophyll content in each leaf. Samples were then stored in a solution of 30% glycerol. The presence of H2 O2 was detected as a reddish-brown colour within the leaf tissue.34 2.8 Analysis of lipid peroxidation Malondialdehyde (MDA) is one of the final products of peroxidation of unsaturated fatty acids found in phospholipids and is responsible for cell membrane damage.35 Lipid peroxidation was measured by determining the MDA content of the ground seedling tissues using a thiobarbituric acid (TBA) reaction as described by Hara et al.36 Briefly, 0.1 g of frozen ground tissues was homogenised in 1 mL of 5 mM potassium phosphate buffer (pH 7). After centrifuging at 4 ∘ C for 15 min at 12 000 × g, an aliquot of the supernatant (900 μL) was mixed with 600 μL of TBA solution containing 10% (w/v) SDS, 20% (w/v) acetic acid, 0.8% (w/v) aqueous TBA and deionised water. The control reaction was a mixture of 900 μL of 5 mM KP buffer and 600 μL of the TBA solution. These mixtures were incubated at 98 ∘ C for 60 min and then cooled to room temperature. Mixtures were centrifuged at 12 000 × g for 15 min at room temperature. The absorbance of the mixture was measured at 535 and 600 nm. The MDA content was calculated from the subtracted absorbance (A 535–600) using a molecular extinction coefficient of 1.56 × 105 M−1 cm−1 . 2.9 Analysis of gene expression using quantitative real-time PCR (QRT-PCR) QRT-PCR was conducted to test the effect of thiamethoxam on the transcript response of ZmCHS, ZmCAD, ZmCAT3, ZmAPX2, ZmSOD2 and ZmGST1 genes to the presence of neighbouring weeds. Total RNA from each treatment was isolated from the different seedling tissues using Tripure reagent (Sigma-Aldrich, St Louis, MO).
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2.5 Analysis of DPPH radical scavenging activity The antioxidant capacity of the sample extracts was tested by evaluation of the free radical scavenging effect on the DPPH radical, according to the method of Abe et al.32 Briefly, 0.1 g of frozen ground tissue from each seedling tissue was extracted with 1 mL of 99.5% methanol. The extract was then centrifuged at 12 000 × g for 15 min. An aliquot (100 μL) of the methanolic extract was mixed with 400 μL of absolute ethanol, 250 μL of 0.5 mM DPPH and 500 μL of 100 mM acetate buffer (pH 5.5). The mixture was vortex mixed and kept in the dark for 30 min. The absorbance of the solution was measured at 517 nm against blanks of DPPH solution that had been prepared similarly but without plant tissue. Results were expressed as the percentage of inhibition of the
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Table 1. Primers sequences used in performing QRT-PCR Primer name ZmTub2 ZmCHS ZmCAD ZmGST1 ZmSOD2 ZmAPX2 ZmCAT3
Forward primer sequence
Reverse primer sequence
5′ -GCGCCTGTCTGTTGACTATGG-3′ 5′ -CTACTTCCGGATCACCAAGAGC-3′ 5′ -ACACAGGCCCTGAAGATGTGGT-3′ 5′ -GGTTCCAGCTCTGCAGGATG-3′ 5′ -CAACGGCTGCATGTCGACT-3′ 5′ -TCGACAAGGCCAAGCGTAAG-3′ 5′ -CCCAAAGGTCAGCCAGGAG-3′
Accession number
5′ -GGGATGGGTACACGGTGAAA-3′ 5′ -CGTCAGGTGCATGTAACGCTT-3′ 5′ -AGGATACTTTGAAGCCCCGAGG-3′ 5′ -ATTGCTCGTGATTCGAAGAGG-3′ 5′ -TGCTCCTTGCCAACAGGATT-3′ 5′ -CGCAATTCTTCTCGGCGAT-3′ 5′ -TTGGCGAGGAGGTCTATCCA-3′
gi|195610153 gi|226505455 gi|162460803 gi|162460885 gi|162462123 gi|162457708 gi|168436
Table 2. Phenotypic parameters of maize seedlings at the fourth-leaf-tip stage as influenced by thiamethoxam applied as a seed treatment. WF and W refer to maize seedlings originating from untreated seeds and grown under weed-free and weedy conditions respectively. TWF and TW refer to maize seedlings originating from thiamethoxam-treated seeds and grown under weed-free and weedy conditions respectively. Treatment means (±SE) followed by different letters indicate significance at P ≤ 0.05 Treatment Phenotypic parameters Second-leaf collar height (cm) Stem diameter (mm) Shoot biomass (g) Root biomass (g) Crown-root number Crown-root length
WF
W
8.6 ± 0.06 a 4.9 ± 0.039 a 0.14 ± 0.004 a 0.10 ± 0.002 a 4.8 ± 0.20 a 91.3 ± 3.41a
To eliminate any residual genomic DNA, total RNA was treated with RQ1 RNase-free DNase (Promega, Madiso, WI). The first-strand cDNA was synthesised from total RNA using the Reverse Transcription System kit (Quanta, Gaithersburg, MD). Primer Express 2.0 software (Applied Biosystems, Carlsbad, CA) was used to design the primers for the target genes (see the description of primer sequences in Table 1). Relative quantification (RQ) values for each target gene relative to the internal control tubulin was calculated by the 2−ΔΔCt method.37 2.10 Light measurements Light quantity (PPFD) and light quality (R:FR) were measured during the experimental period. Incoming PPFD was quantified 10 cm above the maize seedlings using a point quantum radiometer (LI-190SA; LI-COR Biosciences, Lincoln, NE) with a cosine-corrected sensor on a fibre-optic cable. The R:FR of incoming irradiance and the amount reflected upwards from the base of the seedlings to the underside of the first leaves was measured using an R:FR sensor (SKR 110; Skye Instruments Ltd, Llandrindod Wells, Powys, UK). The R:FR reflected from the surface of the different treatments was determined by positioning the sensor downwards, 10 cm above the treatment surface and parallel to the plant at a distance of 10 cm at four equally spaced positions around the circumference of the seedlings. These measurements were recorded for each treatment immediately after planting the maize seeds. The R:FR measured from incoming irradiance did not differ among treatments (R:FR ± SE = 1.56 ± 0.04). The reflected R:FR differed between weed-free and weedy treatments. The R:FR in the weed-free treatments containing only Turface was 1.42 ± 0.03, and in the weedy treatment it was 0.30 ± 0.02.
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2.11 Experimental design and statistical analyses All experiments were conducted under controlled environmental conditions and designed as a completely randomised block.
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9.9 ± 0.10 b 4.4 ± 0.038 b 0.12 ± 0.003 b 0.08 ± 0.005 b 3.6 ± 0.05 b 77.5 ± 2.11 b
TWF
TW
8.9 ± 0.03 a 4.8 ± 0.076 a 0.14 ± 0.003 a 0.10 ± 0.004 a 4.82 ± 0.09 a 93.6 ± 1.63 a
8.8 ± 0.05 a 4.7 ± 0.040 a 0.14 ± 0.004 a 0.09 ± 0.006 a 4.5 ± 0.12 a 91.2 ± 2.33 a
In this experiment, replications were defined as growth cabinet environments in time and were combined for analysis. Each cabinet was divided into two parts using a white, opaque plastic divider to minimise any potential contamination of the R:FR between weed-free and weedy treatments. All experiments were repeated 5 times and, within each replication, individual treatments were re-randomised within each chamber. All statistical analyses were performed using SAS 9.1 (SAS Institute, Cary, NC) with a type 1 error rate set at P ≤ 0.05. The Shapiro–Wilk statistic was used to test for the assumption of normality. No transformation was required prior to the statistical analysis. Significant differences among treatments were analysed using Tukey’s honestly significant difference test.
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RESULTS
3.1 Thiamethoxam as a seed treatment overcoming the morphological expression of shade avoidance in maize seedlings when exposed to neighbouring weeds Maize seedlings originating from untreated seeds expressed typical morphological shade avoidance responses when exposed to neighbouring weeds. An increase in seedling stem height and a reduction in stem diameter, shoot and root biomass and crown-root number and length were observed (Table 2). In contrast, maize seedlings originating from treated seeds grown in either weed-free or weedy conditions did not differ in the morphological parameters as described previously. An increase in stem height was anticipated under weedy conditions for seedlings arising from the untreated seeds. In this experiment, however, height to the second-leaf collar of seedlings originating from thiamethoxam-treated seeds did not differ between treatments: 8.9 ± 0.03 cm in weed-free treatment versus 8.8 ± 0.05 cm in the weedy treatment.
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Thiamethoxam alters maize seedlings response to weeds 3.2 Thiamethoxam as a seed treatment enabling maize seedlings to maintain the total phenolics content and DPPH radical scavenging activity in spite of the presence of neighbouring weeds Total phenolics content was higher in the first-leaf, stem and crown-root tissues of maize seedlings originating from seeds treated with thiamethoxam in comparison with seedlings originating from untreated seeds under both weed-free and weedy conditions (Fig. 1). Under weed-free conditions, the total phenolics content in the first leaf of seedlings originating from treated and untreated seeds was 261 ± 2.4 μg g−1 compared with 253 ± 1.3 μg g−1 respectively (Fig. 1A). In addition, the presence of neighbouring weeds reduced total phenolics content in the first leaf, stem and crown roots of seedlings originating from untreated seeds. In the stem, the total phenolics content was reduced from 59 ± 0.9 μg g−1 in the weed-free treatment to 54 ± 0.4 μg g−1 in the weedy treatment. This contrasted with the results obtained from seedlings originating from thiamethoxam-treated seeds. There was no difference in the total phenolics content in the first leaf, stem or crown roots of seedlings grown under weed-free or weedy conditions. The crown-root total phenolics content was 52 ± 3.03 μg g−1 under weed-free conditions versus 51 ± 2.4 μg g−1 under weedy conditions. An identical response was observed in the DPPH radical scavenging activity in the same tissues. Thus, the DPPH radical scavenging activity was higher in the first-leaf, stem and crown-root tissue of maize seedlings originating from seeds treated with thiamethoxam in comparison with seedlings originating from untreated seeds under both weed-free and weedy conditions (Fig. 2). In contrast, the presence of neighbouring weeds reduced DPPH radical scavenging activity in the first leaf, stem and crown roots of seedlings originating from untreated seeds. The DPPH radical scavenging activity in the first leaf was reduced from 55 ± 1.6 in the weed-free treatment to 50 ± 0.6 in the weedy treatment. This contrasted with the results obtained from seedlings originating from thiamethoxam-treated seeds. There was no difference in DPPH radical scavenging activity in the first leaf, stem and crown roots of seedlings grown under weed-free or weedy conditions.
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Figure 1. Total phenolics content (A) in the first-leaf tissue, (B) in the stem tissue and (C) in the crown-root tissue of maize seedlings, as influenced by the above-ground neighbouring weeds at the fourth-leaf-tip stage of maize development. WF and W refer to maize seedlings originating from untreated seeds and grown under weed-free and weedy conditions respectively. TWF and TW refer to maize seedlings originating from thiamethoxam-treated seeds and grown under weed-free and weedy conditions respectively.
conditions did not differ from the control for either untreated or treated seeds, and were non-detectable for any treatments in the crown roots (data not shown). In order to explore the molecular mechanism contributing to these findings, a QRT-PCR was conducted to investigate the transcription level of the chalcon synthase (ZmCHS), a key gene in the anthocyanin pathway, and cinnamyl alcohol dehydrogenase (ZmCAD), a key gene involved in lignin biosynthesis. The results of QRT-PCR confirmed previous findings. The stem tissue from seedlings originating from treated seed had higher ZmCHS and ZmCAD transcript levels in both weedy and weed-free treatments compared with those seedlings originating from untreated seeds and grown under weed-free conditions. Moreover, exposure to neighbouring weeds caused a reduction in the transcript level of ZmCHS in the stem tissue of maize seedlings originating from untreated seed from onefold in the weed-free treatment to 0.44-fold in the weedy treatment (Fig. 3B). In contrast, no reduction was observed in the transcription level of ZmCHS in the stem tissue of seedlings originating from thiamethoxam-treated seed and exposed to neighbouring weeds in comparison with
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3.3 Thiamethoxam as a seed treatment enabling maize seedling stem tissues to maintain the anthocyanin and lignin contents in spite of the presence of neighbouring weeds The concentrations of anthocyanins and lignin were higher in the stem tissues of maize seedlings originating from seeds treated with thiamethoxam in comparison with seedlings originating from untreated seeds under both weed-free and weedy conditions. For example, under weed-free conditions, the stem anthocyanin content in seedlings originating from treated seed was 0.25 ± 0.01 compared with 0.2 ± 0.01 in untreated seed (Fig. 3A). In addition, the presence of neighbouring weeds reduced the stem anthocyanin content and increased the lignin content in seedlings originating from untreated seeds compared with those under weed-free conditions. The stem anthocyanin content in seedlings originating from untreated seeds was reduced from 0.2 ± 0.01 in the weed-free treatment to 0.14 ± 0.01 in the weedy treatment. This contrasted with the results obtained from seedlings originating from thiamethoxam-treated seeds. No differences were observed in either the stem anthocyanin content or the lignin content between seedlings grown under weed-free or weedy conditions. For example, the stem lignin content was 1.4 ± 0.07 under weed-free conditions versus 1.4 ± 0.06 under weedy conditions. The anthocyanin and lignin contents in the first leaf under weedy
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Figure 2. DPPH radical scavenging activity (A) in the first-leaf tissue, (B) in the stem tissue and (C) in the crown-root tissue of maize seedlings, as influenced by the above-ground neighbouring weeds at the fourth-leaf-tip stage of maize development. WF and W refer to maize seedlings originating from untreated seeds and grown under weed-free and weedy conditions respectively. TWF and TW refer to maize seedlings originating from thiamethoxam-treated seeds and grown under weed-free and weedy conditions respectively.
the weed-free control. The stem tissue from seedlings originating from treated seed had higher ZmCHS transcript levels (i.e. 2.54- and 2.63-fold) in the weedy and weed-free treatment, respectively, compared with the onefold higher level in seedlings originating from untreated seeds and grown under weed-free conditions. An identical response was observed in the tissue of the first leaf and crown roots (Fig. 3C). Additionally, exposure to neighbouring weeds caused an increase in the ZmCAD transcript level from onefold in weed-free conditions to 4.7-fold in the stem tissue of seedlings originating from untreated seed. No increase, however, was observed in the transcription level of ZmCAD in the stem tissue of seedlings originating from thiamethoxam-treated seed in the weedy treatment compared with the weed-free treatment. An identical response was also observed in the tissues of the first leaf and crown roots.
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3.4 Thiamethoxam as a seed treatment enabling maize seedlings to reduce the H2 O2 and lipid peroxidation contents in spite of the presence of neighbouring weeds An increase in the H2 O2 and MDA contents was also observed in the first-leaf and crown-root tissue of maize seedlings originating from untreated seeds under weedy conditions (Figs 4 and 5). However, no differences were observed in the H2 O2 or MDA content
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Figure 3. Effect of thiamethoxam as a seed treatment on anthocyanin and lignin biosynthesis in maize seedlings. (A) Anthocyanin and lignin contents in maize-seedling stem tissue, as influenced by above-ground neighbouring weeds at the fourth-leaf-tip stage of maize development. (B) and (C) gives the results of QRT-PCR analysis of the transcript level of chalcon synthesis (ZmCHS) and cinnamyl alcohol dehydrogenase (ZmCAD) genes, respectively, as influenced by the above-ground neighbouring weeds at the fourth-leaf-tip stage of maize development. Data expressed relative to weed-free treatment. WF and W refer to maize seedlings originating from untreated seeds and grown under weed-free and weedy conditions respectively. TWF and TW refer to maize seedlings originating from thiamethoxam-treated seeds and grown under weed-free and weedy conditions respectively.
between seedlings originating from treated seeds grown under weed-free or weedy conditions (Figs 4 and 5). For example, in the weed-free treatment, the H2 O2 concentration of the first-leaf tissue was 0.503 ± 0.016 nM versus 0.721 ± 0.017 nM in the weedy treatment (Fig. 4A). This increase in the first-leaf H2 O2 content was visualised and confirmed by DAB staining in seedlings originating from untreated seeds (Fig. 4C). In contrast, the H2 O2 concentration in the first leaf of seedlings originating from treated seeds was 0.487 ± 0.016 nM in the weed-free treatment compared with 0.491 ± 0.034 nM in weedy treatment. An identical response was observed for the MDA content in the same tissues (Fig. 5). For example, in the weed-free treatment, the MDA content of the first-leaf tissue originating from untreated seeds was 222.1 ± 2.9 versus 255.9 ± 7.4 in the weedy treatment (Fig. 5A). In contrast, the
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Figure 4. Hydrogen peroxide (H2 O2 ) content in (A) the first-leaf tissue and (B) the crown-root tissue of maize seedlings, as influenced by the above-ground neighbouring weeds at the fourth-leaf-tip stage of maize development. (C) H2 O2 detection in the first leaf of maize using the DAB staining method. WF and W refer to maize seedlings originating from untreated seeds and grown under weed-free and weedy conditions respectively. TWF and TW refer to maize seedlings originating from thiamethoxam-treated seeds and grown under weed-free and weedy conditions respectively.
MDA content in the first leaf of seedlings originating from treated seeds was 218.8 ± 4.8 nM in the weed-free treatment compared with 216.4 ± 3.5 nM in the weedy treatment.
Figure 5. Malondialdehyde (MDA) content in (A) the first-leaf tissue and (B) the crown-root tissue of maize seedlings, as influenced by the above-ground neighbouring weeds at the fourth-leaf-tip stage of maize development. WF and W refer to maize seedlings originating from untreated seeds and grown under weed-free and weedy conditions respectively. TWF and TW refer to maize seedlings originating from thiamethoxam-treated seeds and grown under weed-free and weedy conditions respectively.
treated seeds. Under weedy conditions, the transcript level of ZmCAT3 was upregulated to 3.1-fold in the crown roots of maize seedlings originating from treated seeds compared with 2.1-fold upregulation under weed-free conditions. A similar response was observed in the transcript levels of ZmSOD and ZmAPX genes in the crown roots; this response, however, did not differ for the transcript level of ZmGST in crown-root tissue in seedlings emerging from treated seeds.
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DISCUSSION
In this study, maize seedlings originating from seeds treated with thiamethoxam did not express typical shade avoidance characteristics when grown in the presence of neighbouring weeds. This lack of a shade avoidance response was observed in terms of morphological, physiological and molecular processes. This is the first report to identify the mode of action of thiamethoxam with the physiological mechanisms of early crop and weed competition. These results suggest that thiamethoxam enabled seedlings to maintain their antioxidant protective system to avoid damage caused by oxidative stress in spite of the presence of neighbouring weeds. Thiamethoxam enhanced root development and seedling vigour even under light quality stress conditions (i.e. low R:FR)
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3.5 Thiamethoxam activation of the transcription of H2 O2 -scavenging enzyme genes The first-leaf and crown-root tissues of maize seedlings originating from thiamethoxam-treated seeds had higher transcript levels of ZmCAT, ZmSOD2, ZmAPX and ZmGST genes compared with seedlings emerging from untreated seeds (Fig. 6). This increase in gene activity was consistent under both weed-free and weedy conditions. For example, under weed-free conditions the transcript level of ZmCAT3 in the first leaf was onefold in seedlings from untreated seeds compared with 3.1-fold in seedlings from treated seeds (Fig. 6A). In addition, the presence of neighbouring weeds increased the transcript level of ZmCAT3 within the crown-root tissues of maize seedlings originating from both untreated and
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(A) (C)
(B) (D)
Figure 6. QRT-PCR analysis of the transcript level of (A) catalase (ZmCAT3), (B) superoxide dismutase (ZmSOD2), (C) ascorbate peroxidase (ZmAPX2) and (D) glutathione S-transferase 1 (ZmGST1), as influenced by the above-ground neighbouring weeds at the fourth-leaf-tip stage of maize development. Data presented relative to weed-free treatment. WF and W refer to maize seedlings originating from untreated seeds and grown under weed-free and weedy conditions respectively. TWF and TW refer to maize seedlings originating from thiamethoxam-treated seeds and grown under weed-free and weedy conditions respectively.
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created by the presence of neighbouring weeds. Under stress-free conditions, an increase in seed germination, greater root development and enhancement in seedling vigour have been reported in soybean seedlings originating from thiamethoxam-treated seeds.5,6,38 In the presence of stress caused by salt and aluminium, Cataneo et al.6 observed an increase in seed germination in the presence of salt and aluminium stress with seeds treated with thiamethoxam. This reported acceleration in germination of treated seed correlated with an increase in peroxidase activity. This enzyme is capable of scavenging for H2 O2 in order to prevent oxidative stress. Physiologically, thiamethoxam reduced H2 O2 accumulation and the subsequent damage to cells by reducing peroxidation of lipids. These findings were associated with the upregulation of scavenging genes and the maintenance of phenolic and anthocyanin levels in maize seedlings originating from treated seeds even when exposed to neighbouring weeds. The enhanced production of ROS during environmental stresses can cause damage to biomolecules such as lipids, proteins and DNA. These reactions can alter essential membrane properties such as ion transport, inhibition of protein synthesis and loss of enzyme activity, resulting in cell death.16 Avoiding H2 O2 accumulation and the subsequent enhancement in the antioxidant system comprise a mechanism by which a plant is able to balance the natural antioxidant and ROS production when exposed to abiotic and biotic stress. The balance between the ROS-producing and ROS-scavenging mechanisms can be changed, depending upon the physiological condition of
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the plant and the integration of different environmental, developmental and biochemical stimuli.13 Efficient scavenging of ROS produced as a result of environmental stress requires the action of several non-enzymatic as well as enzymatic antioxidants present in the tissues.16 Stimulating the antioxidant system will promote seedling vigour under stress conditions.11,39 The overexpression of free-radical-scavenging enzymes such as SOD, CAT, APX, GR, DHAR, GST and GPX has been reported to improve stress tolerance in various crop plants.13 Gill and Tuteja14 suggested that plants with the ability to scavenge and/or control the level of cellular free radicals may be better adapted for survival under harsh environmental conditions. Additionally, phenolics including anthocyanins serve as potent non-enzymatic antioxidants within the cell. Phenolics interact with cell signalling and influence gene expression, resulting in modulation of specific enzymatic activities that drive the intracellular responses against oxidative stress.40 For example, anthocyanin is well known for its scavenging activity and its ability to remove free radicals from plant tissues, thereby increasing plant tolerance to subsequent stress conditions.12 Moreover, phenolics influence plant growth and development by modulating processes from germination and cell elongation to senescence.16,41 Maintenance of a high level of antioxidant capacity is an important mechanism by which maize seedlings overcome the negative physiological effects resulting from exposure to low R:FR reflected from neighbouring weeds. Thiamethoxam is known to
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Thiamethoxam alters maize seedlings response to weeds induce antioxidant activity within treated plants by increasing the radical-scavenging antioxidant activity of DPPH measured within the phenolic extract of maize seedling leaves2 or by enhancing ascorbic and salicylic acid levels.9,10 Thus, the maintenance of high antioxidant activity by thiamethoxam would account for the observed enhancement in seedling vigour, reported previously.5,6 Low R:FR reflected from neighbouring weeds has been reported previously to modulate the phenylpropanoid pathway, resulting in a reduction in the anthocyanin content and an enhancement in lignin synthesis in maize seedlings.24 In this study, thiamethoxam was able to maintain the expression of CHS and CAD and consequently maintain the anthocyanin and lignin levels even under weedy conditions. This finding is supported by the fact that thiamethoxam was reported recently to enhance the phenyl ammonia layase (PAL) activity, a key enzyme in the phenylpropoanoid pathway, the site of anthocyanin and lignin biosynthesis.5,7
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CONCLUSIONS
Under non-limiting resource conditions, thiamethoxam was found to enhance seedling vigour and to overcome the expression of typical shade avoidance characteristics caused by neighbouring weeds. The physiological mechanisms by which these results were achieved were attributed to an enhancement in seedling vegetative growth, the maintenance of the phenolics content and the activation of scavenging genes, which reduced the accumulation of H2 O2 in plant tissues. This reduction in H2 O2 content within plant tissues reduced the extent to which cellular damage occurred. This would reduce the energy expended for cellular repair and allow for this energy to be allocated to growth and maintenance of plant tissues. In addition, this work has several implications for the role of seed treatments in agriculture. Normally, seed treatments are thought of only in terms of insect and disease control. The results of the present study, however, suggest the possibility of exploring entirely new chemistries and new modes of action to enhance free radical scavenging and activate genes involved in the antioxidant defence system. This new role for seed treatments may be critical in the development of crop hybrids and cultivars that are more stress tolerant to weed competition.
ACKNOWLEDGEMENTS The financial support of the Natural Science and Engineering Research Council of Canada, File CRDPJ 425128-11, Syngenta Crop Protection and the Ontario Ministry of Agriculture, Food and Rural Affairs is gratefully acknowledged. The authors would also like to express their appreciation to Harold Wright from Syngenta and to Dr Steven Rothstein from the Department of Molecular and Cellular Biology, University of Guelph, for their contributions throughout this study.
REFERENCES
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4 Moser SE and Obrycki JJ, Non-target effects of neonicotinoid seed treatments; mortality of coccinellid larvae related to zoophytophagy. Biol Control 51:487–492 (2009). 5 Macedo WR and Castro CPR, Thiamethoxam: molecule moderator of growth, metabolism and production of spring wheat. Pestic Biochem Physiol 100:299–304 (2011). 6 Cataneo AC, Nunes JC, Ferreira LC, Corniani N, Carvalho JC and Sanine MS, Enhancement of soybean seed vigour as affected by thiamethoxam under stress conditions, in Soybean Physiology and Biochemistry, 1st edition, ed. by El-Shemy HA. InTech, Rijeka, Croatia, pp. 231–274 (2011). 7 Macedo WR, Araújo DK and Castro PR, Unravelling the physiologic and metabolic action of thiamethoxam on rice plants. Pestic Biochem Physiol 107:244–249 (2013). 8 Szczepaniec A, Raupp MJ, Parker RD, Kerns D and Eubanks MD, Neonicotinoid insecticides alter induced defenses and increase susceptibility to spider mites in distantly related crop plants. PloS ONE 8:1–11 (2013). 9 Ford KA, Casida JE, Chardran D, Gulevich AG, Okrent RA, Durkin KA, et al, Neonicotinoid insecticides induce salicylate-associated plant defense responses. Proc Natl Acad Sci USA 107:17 527–17 532 (2010). 10 Bi JL, Tuan SJ and Toscano NC, Impact of greenhouse whitefly management on strawberry fruit quality. Insect Sci 14:151–156 (2007). 11 Umair A, Ali S, Tareen MJ, Ali I and Tareen MN, Effects of seed priming on the antioxidant enzymes activity of mungbean (Vigna radiata) seedlings. Pak J Nutr 11:140–144 (2012). 12 Gould K, Nature’s swiss army knife: the diverse protective roles of anthocyanins in leaves. J Biomed Biotechnol 5:314–320 (2004). 13 Mittler R, Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7:405–410 (2002). 14 Gill SS and Tuteja N, Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48:909–930 (2010). 15 Perveen S, Anis M and Aref IM, Lipid peroxidation, H2 O2 content, and antioxidants during acclimatization of Abrus precatorius to ex vitro conditions. Biol Plant 57:417–424 (2013). 16 Sharma P, Jha AB, Dubey RS and Pessarakli M, Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. J Bot 2012:1–26 (2012). 17 Michalak A, Phenolic compounds and their antioxidant activity in plants growing under heavy metal stress. Pol J Environ Stud 15:523–530 (2006). ´ ´ 18 Janas KM, Amarowicz R, Zielinska-Tomaszewska J, Kosinska A and Posmyk MM, Induction of phenolic compounds in two dark-grown lentil cultivars with different tolerance to copper ions. Acta Physiol Plant 31:587–595 (2009). 19 Lois R and Buchanan BB, Severe sensitivity to ultraviolet radiation in an Arabidopsis mutant deficient in flavonoid accumulation. II. Mechanisms of UV-resistance in Arabidopsis. Planta 194:504–509 (1994). 20 Klessig DF, Durner J, Noad R, Navarre DA, Wendehenne D, Kumar D, et al, Nitric oxide and salicylic acid signaling in plant defense. Proc Natl Acad Sci USA 97:8849–8855 (2000). 21 Vogt T, Phenylpropanoid biosynthesis. Mol Plant 3:2–20 (2010). 22 Besseau S, Hoffman L, Geoffroy P, Lapierre C, Pollet B and Legrand M, Flavonoid accumulation in arabidopsis repressed in lignin synthesis affects auxin transport and plant growth. Plant Cell 19:148–162 (2007). 23 Peng M, Hudson D, Schofield A, Tsao R, Yang R, Gu H, et al, Adaptation of arabidopsis to nitrogen limitation involves induction of anthocyanin synthesis which is controlled by the NLA gene. J Exp Bot 59:2933–2944 (2008). 24 Afifi M and Swanton CJ, Early physiological mechanisms of weed competition. Weed Sci 60:542–551 (2012). 25 De Vos RCH, Vonk MJ, Vooijs R and Schat H, Glutathione depletion due to copper-induced phytochelatin synthesis causes oxidative stress in Silene cucubalus. Plant Physiol 98:853–858 (1992). 26 Tanou G, Molassiotis A and Diamantidis G, Induction of reactive oxygen species and necrotic death-like destruction in strawberry leaves by salinity. Environ Exp Bot 65:270–281 (2009). 27 Mishra S, Jha AB and Dubey RS, Arsenite treatment induces oxidative stress, upregulates antioxidant system, and causes phytochelatin synthesis in rice seedlings. Protoplasma 248:565–577 (2011). 28 Afifi M and Swanton CJ, Maize seed and stem roots differ in response to neighbouring weeds. Weed Res 51:442–450 (2011).
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1 Maienfisch P, Angst M, Brandl F, Fischer W, Hofer D, Kayser H, et al, Chemistry and biology of thiamethoxam: a second-generation neonicotinoid. Pest Manag Sci 57:906–913 (2001). 2 Horii A, McCue P and Shetty K, Enhancement of seed vigour following insecticide and phenolic elicitor treatment. Bioresour Technol 98:623–632 (2007). 3 Elbert A, Haas M, Springer B, Thielert W and Nauen R, Applied aspects of neonicotinoid uses in crop protection. Pest Manag Sci 64:1099–1105 (2008).
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www.soci.org 29 Singleton VL, Orthofer R and Lamuela-Raventos RM, Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin–Ciocalteu reagent. Meth Enzymol 299:152–178 (1999). 30 Neff MM and Chory J, Genetic interactions between phytochrome A, phytochrome B, and cryptochrome 1 during arabidopsis development. Plant Physiol 118:27–35 (1998). 31 Bruce RJ and West CA, Elicitation of lignin biosynthesis and isoperoxidase activity by pectic fragments in suspension cultures of castor bean. Plant Physiol 91:889–897 (1989). 32 Abe N, Murata T and Hirota A, Novel 1,1-diphenyl-2-picryhydrazyl-radical scavengers, bisorbicillin and demethyltrichodimerol, from a fungus. Biosci Biotechnol Biochem 62:661–662 (1998). 33 Patterson BD, Macrae EA and Ferguson IB, Estimation of hydrogen peroxide in plant extracts using titanium (IV). Analyt Biochem 139:487–492 (1984). 34 Thordal-Cheistensen H, Zhang Z, Wei Y and Collinge DB, Subcellular localization of H2 O2 in plants. H2 O2 accumulation in papillae and hypersensitive response during the barley–powdery mildew interaction. Plant J 11:1187–1194 (1997). 35 Halliwell B and Gutteridge JMC, Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J 219:1–14 (1984).
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36 Hara M, Terashima S, Fukaya T and Kuboi T, Enhancement of cold tolerance and inhibition of lipid peroxidation by citrus dehydrin in transgenic tobacco. Planta 217:290–298 (2003). 37 Livak KJ and Schmittgen TD, Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods 25:402–408 (2001). 38 Dan LGM, Dan HA, Braccini AL, Barroso AL, Ricci TT, Piccinin GG, et al, Insecticide treatment and physiological quality of seeds, in Insecticides – Advances in Integrated Pest Management, ed. by Perveen F, 1st edition. InTech, Rijeka, Croatia, pp. 327–342 (2012). 39 Yangui T, Sayadi S, Chakroun H and Dhouib A, Effect of hydroxytyrosol-rich preparations on phenolic-linked antioxidant activity of seeds. Eng Life Sci 11:511–516 (2011). 40 Masella R, Di Benedetto R, Vari R, Filesi C and Giovannini C, Novel mechanisms of natural antioxidant compounds in biological systems: involvement of glutathione and glutathione related enzymes. J Nutr Biochem 16:577–586 (2005). 41 Krogmeier MJ and Bremner JM, Effects of phenolic acids on seed germination and seedling growth in soil. Biol Fert Soils 8:116–122 (1989).
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Accepted article published: 2 April 2014
Published online in Wiley Online Library: 15 May 2014
(wileyonlinelibrary.com) DOI 10.1002/ps.3789
Thiamethoxam as a seed treatment alters the physiological response of maize (Zea mays) seedlings to neighbouring weeds Maha Afifi, Elizabeth Lee, Lewis Lukens and Clarence Swanton* Abstract BACKGROUND: Thiamethoxam is a broad-spectrum neonicotinoid insecticide that, when applied to seed, has been observed to enhance seedling vigour under environmental stress conditions. Stress created by the presence of neighbouring weeds is known to trigger the accumulation of hydrogen peroxide (H2 O2 ) in maize seedling tissue. No previous work has explored the effect of thiamethoxam as a seed treatment on the physiological response of maize seedlings emerging in the presence of neighbouring weeds. RESULTS: Thiamethoxam was found to enhance seedling vigour and to overcome the expression of typical shade avoidance characteristics in the presence of neighbouring weeds. These results were attributed to maintenance of the total phenolics content, 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activity and anthocyanin and lignin contents. These findings were also associated with the activation of scavenging genes, which reduced the accumulation of H2 O2 and the subsequent damage caused by lipid peroxidation in maize seedlings originating from treated seeds even when exposed to neighbouring weeds. CONCLUSIONS: These results suggest the possibility of exploring new chemistries and modes of action as novel seed treatments to upregulate free radical scavenging genes and to maintain the antioxidant system within plants. Such an approach may provide an opportunity to enhance crop competitiveness with weeds. © 2014 Society of Chemical Industry Keywords: R:FR; corn; morphology; DPPH radical scavenging; anthocyanin; lignin; hydrogen peroxide; free radicals; lipid peroxidation; scavenging genes; antioxidant system
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INTRODUCTION
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Thiamethoxam has been reported to increase the phenolics content and to induce the antioxidant capacity of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging in maize.2 Thiamethoxam also altered expression of genes involved in plant defences and regulated by jasmonic acid (JA), salicylic acid (SA) or both pathways against spider mites in cotton, corn and tomato.8 In Arabidopsis, Ford et al.9 reported that thiamethoxam elevated the level of SA within treated plants. SA is an antioxidant that plays an important role in the defence against plant pathogens and is also known to modulate the response of plants to abiotic and biotic stresses. Bi et al.10 tested thiamethoxam for the control of whitefly, a serious pest of strawberry. They found that thiamethoxam maintained soluble solids and glucose levels within the fruit. They also reported that thiamethoxam increased ascorbic acid within the fruit, thereby enhancing antioxidant activity. Increasing the antioxidant activity would account for the observed enhancement in seedling vigour.11 Plant cells have
∗
Correspondence to: Clarence Swanton, Department of Plant Agriculture, Crop Science Building, University of Guelph, 50 Stone Road E., Guelph, ON N1G 2 W1, Canada. E-mail: [email protected] Department of Plant Agriculture, University of Guelph, Guelph, ON, Canada
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Thiamethoxam [3-(2-chloro-1,3-thiazol-5-ylmethyl)-5-methyl-1,3, 5-oxadiazinan-4-ylidene(nitro)amine] is a broad-spectrum neonicotinoid insecticide.1 This compound controls a wide variety of commercially important crop pests with low use rates and flexible application methods.2 It is commercially used either as a foliar spray or as a soil treatment under the trademark Actara™ and as a seed treatment under the trademark Cruiser®.1,3 When this chemistry is applied as a seed treatment, thiamethoxam is distributed systematically throughout the plant, the concentration decreasing with increasing biomass.4 The use of thiamethoxam as a seed treatment reduces human exposure and the potential for off-target movement of this chemistry into the environment.3 Thiamethoxam applied to seed has been observed to increase seed germination, root growth, seedling height and biomass accumulation.2,5 For example, Cataneo et al.6 reported that thiamethoxam promoted seed germination and suggested that this may reduce the time for crop establishment. The use of thiamethoxam was also found to increase root development and to alter the distribution of photoassimilates.5,7 The physiological mechanism by which this germination and growth enhancement occur is not well known. Such physiological changes may be the result of modification of the metabolic pathways within the seedling.
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evolved antioxidative systems that enable the removal of free radicals.12 – 14 This system includes enzymes such as superoxide dismutase, (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and glutathione S-transferase, (GST). In addition, non-enzymatic compounds such as ascorbic acid, phenols and flavonoids such as anthocyanins are known to be involved in antioxidant defence systems. Polyphenols such as flavonoids can chelate transition metal ions, directly scavenge reactive oxygen species (ROS), delay diffusion of free radicals, limit peroxidative reactions and inhibit lipid peroxidation.15 For example, transgenic potato plants with an increased concentration of flavonoid showed improved antioxidant capacity.16 These phenolic compounds produced via the phenylpropanoid pathway perform important functions involved in protecting plant development during periods of environmental stress.17,18 Flavonoids such as anthocyanins protect plants against UV irradiation, while salicylic acid is implicated in plant–pathogen interactions.19,20 Anthocyanins are produced in the phenylpropanoid pathway, in which chalcone synthase (CHS) catalyses the formation of the flavonoids from p-coumaroyl-CoA, which leads to the synthesis of anthocyanin. Within this pathway there are two additional subpathways, both of which are initiated from p-coumaroyl-CoA. In both pathways, cinnamyl alcohol dehydrogenase (CAD) is responsible for the production of lignin.21 Lignin is embedded in the plant cell walls and plays an important role in the mechanical support and transport of water in plants. The balance between lignin and anthocyanin produced by this pathway is influenced by environmental conditions.22 – 24 For example, nitrogen limitation can cause a metabolic flux to occur within the phenylpropanoid pathway, resulting in a diversion of anthocyanin production to lignin in the nla Arabidopsis mutant.23 This diversion of anthocyanins to lignin production will reduce the ability of the crop plant to scavenge for free radicals and protect itself against photooxidative damage.12 Oxidative damage caused by ROS includes the alteration of DNA, and the oxidation of proteins and lipids.25 Hydrogen peroxide (H2 O2 ) is a major ROS component, known as an intercellular systemic signalling mechanism, which can alter plant metabolism but is also capable of inflicting cellular damage.14 Accumulation of H2 O2 increases the possibility of the formation of hydroxyl radicals, which may account for subsequent lipid peroxidation. Levels of lipid peroxidation have been used widely as an indicator of ROS-mediated damage to cell membranes under stressful conditions.26,27 During stressful periods, the avoidance of H2 O2 accumulation is an important plant strategy to enhance metabolic recovery. Plants have several mechanisms that can reduce H2 O2 production during a stressful period. These mechanisms include anatomical adaptations and physiological and molecular changes.13 Plants with the ability to scavenge and/or control the level of cellular H2 O2 will be better adapted to survive.14 Efficient scavenging of ROS requires the action of both non-enzymatic and enzymatic scavenging genes.16 An increase in the transcript level of ZmGST1, ZmSOD2, ZmAPX2 and ZmCAT3 genes and modulation of the phenylpropanoid pathway, resulting in a reduction in anthocyanin content, was observed when maize seedlings were exposed to conditions of low R:FR reflected from above-ground neighbouring weeds.24 Accumulation of H2 O2 and a reduction in antioxidant compounds would invariably reduce seedling growth. In contrast, the available literature suggests that seedling vigour was enhanced when seeds were
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treated with thiamethoxam. No previous work has explored the effect of thiamethoxam as a seed treatment on the physiological response of maize seedlings emerging in the presence of neighbouring weeds. Thus, it was hypothesised that this enhancement in growth may be attributed to a reduction in H2 O2 accumulation in seedlings emerging from seed treated with thiamethoxam. It was further hypothesised that thiamethoxam may elevate the expression of genes involved in the scavenging of H2 O2 and the phenylproponoid pathway.
2
MATERIALS AND METHODS
2.1 Plant material and growth conditions Maize seeds (Zea mays) of the hybrid NK-N29T-GT (Syngenta Crop Protection Canada Inc.) were selected for study in this experiment. Thiamethoxam-treated seed (100 g AI 100 kg−1 ) and untreated seeds were planted to a depth of 2 cm, one seed per cup, in 8 cm diameter, 10 cm tall, 355 mL plastic cups (Dart Container Corporation, Mason, MI). These cups were filled with Turface, a 100% backed calcined clay growth medium (Turface MVP®; Profile Products LLC, Buffalo Grove, IL). These cups were then placed in 8 cm diameter, 18 cm tall pots (1 L of natural cylinder modified to 18 cm; Consolidated Bottle Co., Toronto, ON). These cylindrical pots were further centred within 25 cm diameter, 19 cm tall, 6 L pots (Airlite Plastics Company, Omaha, NE), as described in Afifi and Swanton.28 The area surrounding the cylindrical pot was filled with Turface, herein referred to as the weed-free treatment. The weedy treatment was seeded into this area of Turface with Fiesta III (Lolium perenne L.), 2–3 weeks prior to planting. This perennial ryegrass was used as a model weed species. Ryegrass was watered as required with Hoagland’s nutrient solution and allowed to grow and form a dense canopy. This canopy was trimmed once to a height of 5 cm prior to the initiation of each replicate to ensure that no shading of the maize seedling occurred. This design ensured that there was no physical root contact between either species or contamination from either root exudates or direct competition for incoming light, water or nutrients. Pots were placed within the same growth cabinet (Converon, Winnipeg, Canada) set to 28/20 ∘ C, 16:8 h light:dark regime and 60–65% humidity. Irradiance was supplied by a sliding bank of Sylvania Cool White fluorescent tubes and inside-frost tungsten 40 W bulbs delivering 500 μmol m−2 s−1 of PPFD. Maize seedlings within each pot were also fertigated daily using Hoagland’s solution. Ten seedlings per treatment (replicated 5 times, N = 50) were harvested at the fourth-leaf-tip stage. Maize seedling height to the second-leaf collar (i.e. the distance from the Turface to the youngest visible collar) and stem diameter 2 cm above the first stem node were measured. Roots were then washed under running tap water and separated from the above-ground stem by cutting just above the highest crown root. The number and length of crown roots for each seedling were recorded. Shoots and the entire root system were then bagged separately and dried at 80 ∘ C to constant weight to determine total shoot and root biomass. For the physiological and molecular analysis, an additional ten seedlings per treatment, replicated 5 times (N = 50), were separated into component parts of the first leaf, stem and crown roots. The remaining three leaves were discarded. Only the first leaf was selected for analysis, based on the results from a previous study.24 These samples were then frozen immediately in liquid nitrogen and stored at −80 ∘ C for further analysis.
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Thiamethoxam alters maize seedlings response to weeds 2.2 Analysis of the total phenolics content in the seedling extracts The total phenolics content in the tissue was determined according to the method described by Singleton et al.29 Briefly, 0.1 g of frozen ground tissue from each seedling component was extracted with 1 mL of absolute methanol. The methanolic mixture was then centrifuged at 12 000 × g for 15 min. An aliquot of (500 μL) of the methanolic extract was mixed with 2.5 mL of 10% Folin–Ciocalteu’s-reagent-dissolved 7.5% NaHCO3 . The mixture was then incubated at 45 ∘ C for 30 min. The absorbance of the mixture was measured at 765 nm against a blank of Folin–Ciocalteu’s reagent that had been prepared similarly but without plant tissue. A calibration curve was prepared using a standard solution of gallic acid, and the results were expressed as μg gallic acid equivalent g−1 fresh weight of sample. 2.3 Analysis of anthocyanin content Total anthocyanin was determined in the tissues according to the method described by Neff and Chory.30 Briefly, 0.1 g of frozen ground tissue from each seedling tissue was extracted with 1 mL of methanol and acidified with 0.1% HCl overnight at 4 ∘ C. After centrifuging the supernatant for 5 min at 5000 × g, anthocyanin was separated from chlorophyll by adding 250 mL of chloroform. The total anthocyanin content was determined by subtracting the absorption values (A) measured at 657 nm from the A values measured at 530 nm of the aqueous phase using a spectrophotometer. 2.4 Analysis of lignin content Lignin was extracted and measured from the ground tissues by the method of Bruce and West.31 A quantity of 3 g of each plant tissue was homogenised separately in 10 mL of 99.5% ethanol, and the extract was centrifuged at 10 000 × g for 15 min. The remaining pellet was freeze dried. A quantity of 50 mg of the dry pellet was extracted and placed in a solution of 5 mL of 2 N HCl and 0.5 mL of thioglycolic acid. This sample was heated at 100 ∘ C for 8 h, cooled and centrifuged again at 14 000 × g for 30 min at 4 ∘ C. The pellet was washed once with 2.5 mL of water and then resuspended in 5 mL of 1 N NaOH. The solution was agitated gently at 25 ∘ C for 18 h. After a further centrifugation at 14 000 × g for 30 min, 1 mL of concentrated HCl was added to the supernatant in a clean test tube and left overnight at 4 ∘ C for lignin thioglycolate to precipitate. Following a final centrifugation at 14 000 × g for 30 min, the pellet was dissolved in 1 mL of 1 N NaOH. The lignin content within the solution was expressed as the A values measured at 280 nm using 1 N NaOH as the blank solution.
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DPPH radical, which was calculated according to the following equation: %Inhibition of DPPH =
Acontrol − Asample Acontrol
× 100
where Acontrol is the absorbance reading of DPPH in the solution without extracts and Asample is the absorbance reading of DPPH within the sample solution. 2.6 Analysis of H2 O2 concentration Hydrogen peroxide in the ground tissues was estimated according to the protocol reported by Patterson et al.33 A quantity of 100 mg of frozen ground tissues was homogenised in 200 μL of cold acetone. After centrifuging for 5 min at 10 000 × g, the supernatant was mixed with 20 μL of titanium reagent (2% TiC12 in concentrated HCl). The Ti–H2 O2 complex was precipitated by adding 40 μL of 15 M ammonia solution. This solution was centrifuged as described above, after which the pellet was washed with cold acetone 2 times and then dissolved in 1 mL of 4 N H2 SO4 . The absorbance of the solution was measured at 410 nm against blanks that had been prepared similarly but without plant tissue. 2.7 Detection of H2 O2 using the DAB staining method Leaves of maize seedlings were cut carefully and placed in plastic tubes containing 1 mg mL−1 of 3,3-diaminobenzidine (DAB)-HCl (Sigma-Aldrich, St Louis, MO). Samples were incubated in the DAB solution overnight. Leaf tissues were placed in boiling ethanol (96%) in order to clear the chlorophyll content in each leaf. Samples were then stored in a solution of 30% glycerol. The presence of H2 O2 was detected as a reddish-brown colour within the leaf tissue.34 2.8 Analysis of lipid peroxidation Malondialdehyde (MDA) is one of the final products of peroxidation of unsaturated fatty acids found in phospholipids and is responsible for cell membrane damage.35 Lipid peroxidation was measured by determining the MDA content of the ground seedling tissues using a thiobarbituric acid (TBA) reaction as described by Hara et al.36 Briefly, 0.1 g of frozen ground tissues was homogenised in 1 mL of 5 mM potassium phosphate buffer (pH 7). After centrifuging at 4 ∘ C for 15 min at 12 000 × g, an aliquot of the supernatant (900 μL) was mixed with 600 μL of TBA solution containing 10% (w/v) SDS, 20% (w/v) acetic acid, 0.8% (w/v) aqueous TBA and deionised water. The control reaction was a mixture of 900 μL of 5 mM KP buffer and 600 μL of the TBA solution. These mixtures were incubated at 98 ∘ C for 60 min and then cooled to room temperature. Mixtures were centrifuged at 12 000 × g for 15 min at room temperature. The absorbance of the mixture was measured at 535 and 600 nm. The MDA content was calculated from the subtracted absorbance (A 535–600) using a molecular extinction coefficient of 1.56 × 105 M−1 cm−1 . 2.9 Analysis of gene expression using quantitative real-time PCR (QRT-PCR) QRT-PCR was conducted to test the effect of thiamethoxam on the transcript response of ZmCHS, ZmCAD, ZmCAT3, ZmAPX2, ZmSOD2 and ZmGST1 genes to the presence of neighbouring weeds. Total RNA from each treatment was isolated from the different seedling tissues using Tripure reagent (Sigma-Aldrich, St Louis, MO).
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2.5 Analysis of DPPH radical scavenging activity The antioxidant capacity of the sample extracts was tested by evaluation of the free radical scavenging effect on the DPPH radical, according to the method of Abe et al.32 Briefly, 0.1 g of frozen ground tissue from each seedling tissue was extracted with 1 mL of 99.5% methanol. The extract was then centrifuged at 12 000 × g for 15 min. An aliquot (100 μL) of the methanolic extract was mixed with 400 μL of absolute ethanol, 250 μL of 0.5 mM DPPH and 500 μL of 100 mM acetate buffer (pH 5.5). The mixture was vortex mixed and kept in the dark for 30 min. The absorbance of the solution was measured at 517 nm against blanks of DPPH solution that had been prepared similarly but without plant tissue. Results were expressed as the percentage of inhibition of the
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Table 1. Primers sequences used in performing QRT-PCR Primer name ZmTub2 ZmCHS ZmCAD ZmGST1 ZmSOD2 ZmAPX2 ZmCAT3
Forward primer sequence
Reverse primer sequence
5′ -GCGCCTGTCTGTTGACTATGG-3′ 5′ -CTACTTCCGGATCACCAAGAGC-3′ 5′ -ACACAGGCCCTGAAGATGTGGT-3′ 5′ -GGTTCCAGCTCTGCAGGATG-3′ 5′ -CAACGGCTGCATGTCGACT-3′ 5′ -TCGACAAGGCCAAGCGTAAG-3′ 5′ -CCCAAAGGTCAGCCAGGAG-3′
Accession number
5′ -GGGATGGGTACACGGTGAAA-3′ 5′ -CGTCAGGTGCATGTAACGCTT-3′ 5′ -AGGATACTTTGAAGCCCCGAGG-3′ 5′ -ATTGCTCGTGATTCGAAGAGG-3′ 5′ -TGCTCCTTGCCAACAGGATT-3′ 5′ -CGCAATTCTTCTCGGCGAT-3′ 5′ -TTGGCGAGGAGGTCTATCCA-3′
gi|195610153 gi|226505455 gi|162460803 gi|162460885 gi|162462123 gi|162457708 gi|168436
Table 2. Phenotypic parameters of maize seedlings at the fourth-leaf-tip stage as influenced by thiamethoxam applied as a seed treatment. WF and W refer to maize seedlings originating from untreated seeds and grown under weed-free and weedy conditions respectively. TWF and TW refer to maize seedlings originating from thiamethoxam-treated seeds and grown under weed-free and weedy conditions respectively. Treatment means (±SE) followed by different letters indicate significance at P ≤ 0.05 Treatment Phenotypic parameters Second-leaf collar height (cm) Stem diameter (mm) Shoot biomass (g) Root biomass (g) Crown-root number Crown-root length
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8.6 ± 0.06 a 4.9 ± 0.039 a 0.14 ± 0.004 a 0.10 ± 0.002 a 4.8 ± 0.20 a 91.3 ± 3.41a
To eliminate any residual genomic DNA, total RNA was treated with RQ1 RNase-free DNase (Promega, Madiso, WI). The first-strand cDNA was synthesised from total RNA using the Reverse Transcription System kit (Quanta, Gaithersburg, MD). Primer Express 2.0 software (Applied Biosystems, Carlsbad, CA) was used to design the primers for the target genes (see the description of primer sequences in Table 1). Relative quantification (RQ) values for each target gene relative to the internal control tubulin was calculated by the 2−ΔΔCt method.37 2.10 Light measurements Light quantity (PPFD) and light quality (R:FR) were measured during the experimental period. Incoming PPFD was quantified 10 cm above the maize seedlings using a point quantum radiometer (LI-190SA; LI-COR Biosciences, Lincoln, NE) with a cosine-corrected sensor on a fibre-optic cable. The R:FR of incoming irradiance and the amount reflected upwards from the base of the seedlings to the underside of the first leaves was measured using an R:FR sensor (SKR 110; Skye Instruments Ltd, Llandrindod Wells, Powys, UK). The R:FR reflected from the surface of the different treatments was determined by positioning the sensor downwards, 10 cm above the treatment surface and parallel to the plant at a distance of 10 cm at four equally spaced positions around the circumference of the seedlings. These measurements were recorded for each treatment immediately after planting the maize seeds. The R:FR measured from incoming irradiance did not differ among treatments (R:FR ± SE = 1.56 ± 0.04). The reflected R:FR differed between weed-free and weedy treatments. The R:FR in the weed-free treatments containing only Turface was 1.42 ± 0.03, and in the weedy treatment it was 0.30 ± 0.02.
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2.11 Experimental design and statistical analyses All experiments were conducted under controlled environmental conditions and designed as a completely randomised block.
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9.9 ± 0.10 b 4.4 ± 0.038 b 0.12 ± 0.003 b 0.08 ± 0.005 b 3.6 ± 0.05 b 77.5 ± 2.11 b
TWF
TW
8.9 ± 0.03 a 4.8 ± 0.076 a 0.14 ± 0.003 a 0.10 ± 0.004 a 4.82 ± 0.09 a 93.6 ± 1.63 a
8.8 ± 0.05 a 4.7 ± 0.040 a 0.14 ± 0.004 a 0.09 ± 0.006 a 4.5 ± 0.12 a 91.2 ± 2.33 a
In this experiment, replications were defined as growth cabinet environments in time and were combined for analysis. Each cabinet was divided into two parts using a white, opaque plastic divider to minimise any potential contamination of the R:FR between weed-free and weedy treatments. All experiments were repeated 5 times and, within each replication, individual treatments were re-randomised within each chamber. All statistical analyses were performed using SAS 9.1 (SAS Institute, Cary, NC) with a type 1 error rate set at P ≤ 0.05. The Shapiro–Wilk statistic was used to test for the assumption of normality. No transformation was required prior to the statistical analysis. Significant differences among treatments were analysed using Tukey’s honestly significant difference test.
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RESULTS
3.1 Thiamethoxam as a seed treatment overcoming the morphological expression of shade avoidance in maize seedlings when exposed to neighbouring weeds Maize seedlings originating from untreated seeds expressed typical morphological shade avoidance responses when exposed to neighbouring weeds. An increase in seedling stem height and a reduction in stem diameter, shoot and root biomass and crown-root number and length were observed (Table 2). In contrast, maize seedlings originating from treated seeds grown in either weed-free or weedy conditions did not differ in the morphological parameters as described previously. An increase in stem height was anticipated under weedy conditions for seedlings arising from the untreated seeds. In this experiment, however, height to the second-leaf collar of seedlings originating from thiamethoxam-treated seeds did not differ between treatments: 8.9 ± 0.03 cm in weed-free treatment versus 8.8 ± 0.05 cm in the weedy treatment.
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Thiamethoxam alters maize seedlings response to weeds 3.2 Thiamethoxam as a seed treatment enabling maize seedlings to maintain the total phenolics content and DPPH radical scavenging activity in spite of the presence of neighbouring weeds Total phenolics content was higher in the first-leaf, stem and crown-root tissues of maize seedlings originating from seeds treated with thiamethoxam in comparison with seedlings originating from untreated seeds under both weed-free and weedy conditions (Fig. 1). Under weed-free conditions, the total phenolics content in the first leaf of seedlings originating from treated and untreated seeds was 261 ± 2.4 μg g−1 compared with 253 ± 1.3 μg g−1 respectively (Fig. 1A). In addition, the presence of neighbouring weeds reduced total phenolics content in the first leaf, stem and crown roots of seedlings originating from untreated seeds. In the stem, the total phenolics content was reduced from 59 ± 0.9 μg g−1 in the weed-free treatment to 54 ± 0.4 μg g−1 in the weedy treatment. This contrasted with the results obtained from seedlings originating from thiamethoxam-treated seeds. There was no difference in the total phenolics content in the first leaf, stem or crown roots of seedlings grown under weed-free or weedy conditions. The crown-root total phenolics content was 52 ± 3.03 μg g−1 under weed-free conditions versus 51 ± 2.4 μg g−1 under weedy conditions. An identical response was observed in the DPPH radical scavenging activity in the same tissues. Thus, the DPPH radical scavenging activity was higher in the first-leaf, stem and crown-root tissue of maize seedlings originating from seeds treated with thiamethoxam in comparison with seedlings originating from untreated seeds under both weed-free and weedy conditions (Fig. 2). In contrast, the presence of neighbouring weeds reduced DPPH radical scavenging activity in the first leaf, stem and crown roots of seedlings originating from untreated seeds. The DPPH radical scavenging activity in the first leaf was reduced from 55 ± 1.6 in the weed-free treatment to 50 ± 0.6 in the weedy treatment. This contrasted with the results obtained from seedlings originating from thiamethoxam-treated seeds. There was no difference in DPPH radical scavenging activity in the first leaf, stem and crown roots of seedlings grown under weed-free or weedy conditions.
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(C)
Figure 1. Total phenolics content (A) in the first-leaf tissue, (B) in the stem tissue and (C) in the crown-root tissue of maize seedlings, as influenced by the above-ground neighbouring weeds at the fourth-leaf-tip stage of maize development. WF and W refer to maize seedlings originating from untreated seeds and grown under weed-free and weedy conditions respectively. TWF and TW refer to maize seedlings originating from thiamethoxam-treated seeds and grown under weed-free and weedy conditions respectively.
conditions did not differ from the control for either untreated or treated seeds, and were non-detectable for any treatments in the crown roots (data not shown). In order to explore the molecular mechanism contributing to these findings, a QRT-PCR was conducted to investigate the transcription level of the chalcon synthase (ZmCHS), a key gene in the anthocyanin pathway, and cinnamyl alcohol dehydrogenase (ZmCAD), a key gene involved in lignin biosynthesis. The results of QRT-PCR confirmed previous findings. The stem tissue from seedlings originating from treated seed had higher ZmCHS and ZmCAD transcript levels in both weedy and weed-free treatments compared with those seedlings originating from untreated seeds and grown under weed-free conditions. Moreover, exposure to neighbouring weeds caused a reduction in the transcript level of ZmCHS in the stem tissue of maize seedlings originating from untreated seed from onefold in the weed-free treatment to 0.44-fold in the weedy treatment (Fig. 3B). In contrast, no reduction was observed in the transcription level of ZmCHS in the stem tissue of seedlings originating from thiamethoxam-treated seed and exposed to neighbouring weeds in comparison with
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3.3 Thiamethoxam as a seed treatment enabling maize seedling stem tissues to maintain the anthocyanin and lignin contents in spite of the presence of neighbouring weeds The concentrations of anthocyanins and lignin were higher in the stem tissues of maize seedlings originating from seeds treated with thiamethoxam in comparison with seedlings originating from untreated seeds under both weed-free and weedy conditions. For example, under weed-free conditions, the stem anthocyanin content in seedlings originating from treated seed was 0.25 ± 0.01 compared with 0.2 ± 0.01 in untreated seed (Fig. 3A). In addition, the presence of neighbouring weeds reduced the stem anthocyanin content and increased the lignin content in seedlings originating from untreated seeds compared with those under weed-free conditions. The stem anthocyanin content in seedlings originating from untreated seeds was reduced from 0.2 ± 0.01 in the weed-free treatment to 0.14 ± 0.01 in the weedy treatment. This contrasted with the results obtained from seedlings originating from thiamethoxam-treated seeds. No differences were observed in either the stem anthocyanin content or the lignin content between seedlings grown under weed-free or weedy conditions. For example, the stem lignin content was 1.4 ± 0.07 under weed-free conditions versus 1.4 ± 0.06 under weedy conditions. The anthocyanin and lignin contents in the first leaf under weedy
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Figure 2. DPPH radical scavenging activity (A) in the first-leaf tissue, (B) in the stem tissue and (C) in the crown-root tissue of maize seedlings, as influenced by the above-ground neighbouring weeds at the fourth-leaf-tip stage of maize development. WF and W refer to maize seedlings originating from untreated seeds and grown under weed-free and weedy conditions respectively. TWF and TW refer to maize seedlings originating from thiamethoxam-treated seeds and grown under weed-free and weedy conditions respectively.
the weed-free control. The stem tissue from seedlings originating from treated seed had higher ZmCHS transcript levels (i.e. 2.54- and 2.63-fold) in the weedy and weed-free treatment, respectively, compared with the onefold higher level in seedlings originating from untreated seeds and grown under weed-free conditions. An identical response was observed in the tissue of the first leaf and crown roots (Fig. 3C). Additionally, exposure to neighbouring weeds caused an increase in the ZmCAD transcript level from onefold in weed-free conditions to 4.7-fold in the stem tissue of seedlings originating from untreated seed. No increase, however, was observed in the transcription level of ZmCAD in the stem tissue of seedlings originating from thiamethoxam-treated seed in the weedy treatment compared with the weed-free treatment. An identical response was also observed in the tissues of the first leaf and crown roots.
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3.4 Thiamethoxam as a seed treatment enabling maize seedlings to reduce the H2 O2 and lipid peroxidation contents in spite of the presence of neighbouring weeds An increase in the H2 O2 and MDA contents was also observed in the first-leaf and crown-root tissue of maize seedlings originating from untreated seeds under weedy conditions (Figs 4 and 5). However, no differences were observed in the H2 O2 or MDA content
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Figure 3. Effect of thiamethoxam as a seed treatment on anthocyanin and lignin biosynthesis in maize seedlings. (A) Anthocyanin and lignin contents in maize-seedling stem tissue, as influenced by above-ground neighbouring weeds at the fourth-leaf-tip stage of maize development. (B) and (C) gives the results of QRT-PCR analysis of the transcript level of chalcon synthesis (ZmCHS) and cinnamyl alcohol dehydrogenase (ZmCAD) genes, respectively, as influenced by the above-ground neighbouring weeds at the fourth-leaf-tip stage of maize development. Data expressed relative to weed-free treatment. WF and W refer to maize seedlings originating from untreated seeds and grown under weed-free and weedy conditions respectively. TWF and TW refer to maize seedlings originating from thiamethoxam-treated seeds and grown under weed-free and weedy conditions respectively.
between seedlings originating from treated seeds grown under weed-free or weedy conditions (Figs 4 and 5). For example, in the weed-free treatment, the H2 O2 concentration of the first-leaf tissue was 0.503 ± 0.016 nM versus 0.721 ± 0.017 nM in the weedy treatment (Fig. 4A). This increase in the first-leaf H2 O2 content was visualised and confirmed by DAB staining in seedlings originating from untreated seeds (Fig. 4C). In contrast, the H2 O2 concentration in the first leaf of seedlings originating from treated seeds was 0.487 ± 0.016 nM in the weed-free treatment compared with 0.491 ± 0.034 nM in weedy treatment. An identical response was observed for the MDA content in the same tissues (Fig. 5). For example, in the weed-free treatment, the MDA content of the first-leaf tissue originating from untreated seeds was 222.1 ± 2.9 versus 255.9 ± 7.4 in the weedy treatment (Fig. 5A). In contrast, the
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Figure 4. Hydrogen peroxide (H2 O2 ) content in (A) the first-leaf tissue and (B) the crown-root tissue of maize seedlings, as influenced by the above-ground neighbouring weeds at the fourth-leaf-tip stage of maize development. (C) H2 O2 detection in the first leaf of maize using the DAB staining method. WF and W refer to maize seedlings originating from untreated seeds and grown under weed-free and weedy conditions respectively. TWF and TW refer to maize seedlings originating from thiamethoxam-treated seeds and grown under weed-free and weedy conditions respectively.
MDA content in the first leaf of seedlings originating from treated seeds was 218.8 ± 4.8 nM in the weed-free treatment compared with 216.4 ± 3.5 nM in the weedy treatment.
Figure 5. Malondialdehyde (MDA) content in (A) the first-leaf tissue and (B) the crown-root tissue of maize seedlings, as influenced by the above-ground neighbouring weeds at the fourth-leaf-tip stage of maize development. WF and W refer to maize seedlings originating from untreated seeds and grown under weed-free and weedy conditions respectively. TWF and TW refer to maize seedlings originating from thiamethoxam-treated seeds and grown under weed-free and weedy conditions respectively.
treated seeds. Under weedy conditions, the transcript level of ZmCAT3 was upregulated to 3.1-fold in the crown roots of maize seedlings originating from treated seeds compared with 2.1-fold upregulation under weed-free conditions. A similar response was observed in the transcript levels of ZmSOD and ZmAPX genes in the crown roots; this response, however, did not differ for the transcript level of ZmGST in crown-root tissue in seedlings emerging from treated seeds.
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DISCUSSION
In this study, maize seedlings originating from seeds treated with thiamethoxam did not express typical shade avoidance characteristics when grown in the presence of neighbouring weeds. This lack of a shade avoidance response was observed in terms of morphological, physiological and molecular processes. This is the first report to identify the mode of action of thiamethoxam with the physiological mechanisms of early crop and weed competition. These results suggest that thiamethoxam enabled seedlings to maintain their antioxidant protective system to avoid damage caused by oxidative stress in spite of the presence of neighbouring weeds. Thiamethoxam enhanced root development and seedling vigour even under light quality stress conditions (i.e. low R:FR)
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3.5 Thiamethoxam activation of the transcription of H2 O2 -scavenging enzyme genes The first-leaf and crown-root tissues of maize seedlings originating from thiamethoxam-treated seeds had higher transcript levels of ZmCAT, ZmSOD2, ZmAPX and ZmGST genes compared with seedlings emerging from untreated seeds (Fig. 6). This increase in gene activity was consistent under both weed-free and weedy conditions. For example, under weed-free conditions the transcript level of ZmCAT3 in the first leaf was onefold in seedlings from untreated seeds compared with 3.1-fold in seedlings from treated seeds (Fig. 6A). In addition, the presence of neighbouring weeds increased the transcript level of ZmCAT3 within the crown-root tissues of maize seedlings originating from both untreated and
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Figure 6. QRT-PCR analysis of the transcript level of (A) catalase (ZmCAT3), (B) superoxide dismutase (ZmSOD2), (C) ascorbate peroxidase (ZmAPX2) and (D) glutathione S-transferase 1 (ZmGST1), as influenced by the above-ground neighbouring weeds at the fourth-leaf-tip stage of maize development. Data presented relative to weed-free treatment. WF and W refer to maize seedlings originating from untreated seeds and grown under weed-free and weedy conditions respectively. TWF and TW refer to maize seedlings originating from thiamethoxam-treated seeds and grown under weed-free and weedy conditions respectively.
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created by the presence of neighbouring weeds. Under stress-free conditions, an increase in seed germination, greater root development and enhancement in seedling vigour have been reported in soybean seedlings originating from thiamethoxam-treated seeds.5,6,38 In the presence of stress caused by salt and aluminium, Cataneo et al.6 observed an increase in seed germination in the presence of salt and aluminium stress with seeds treated with thiamethoxam. This reported acceleration in germination of treated seed correlated with an increase in peroxidase activity. This enzyme is capable of scavenging for H2 O2 in order to prevent oxidative stress. Physiologically, thiamethoxam reduced H2 O2 accumulation and the subsequent damage to cells by reducing peroxidation of lipids. These findings were associated with the upregulation of scavenging genes and the maintenance of phenolic and anthocyanin levels in maize seedlings originating from treated seeds even when exposed to neighbouring weeds. The enhanced production of ROS during environmental stresses can cause damage to biomolecules such as lipids, proteins and DNA. These reactions can alter essential membrane properties such as ion transport, inhibition of protein synthesis and loss of enzyme activity, resulting in cell death.16 Avoiding H2 O2 accumulation and the subsequent enhancement in the antioxidant system comprise a mechanism by which a plant is able to balance the natural antioxidant and ROS production when exposed to abiotic and biotic stress. The balance between the ROS-producing and ROS-scavenging mechanisms can be changed, depending upon the physiological condition of
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the plant and the integration of different environmental, developmental and biochemical stimuli.13 Efficient scavenging of ROS produced as a result of environmental stress requires the action of several non-enzymatic as well as enzymatic antioxidants present in the tissues.16 Stimulating the antioxidant system will promote seedling vigour under stress conditions.11,39 The overexpression of free-radical-scavenging enzymes such as SOD, CAT, APX, GR, DHAR, GST and GPX has been reported to improve stress tolerance in various crop plants.13 Gill and Tuteja14 suggested that plants with the ability to scavenge and/or control the level of cellular free radicals may be better adapted for survival under harsh environmental conditions. Additionally, phenolics including anthocyanins serve as potent non-enzymatic antioxidants within the cell. Phenolics interact with cell signalling and influence gene expression, resulting in modulation of specific enzymatic activities that drive the intracellular responses against oxidative stress.40 For example, anthocyanin is well known for its scavenging activity and its ability to remove free radicals from plant tissues, thereby increasing plant tolerance to subsequent stress conditions.12 Moreover, phenolics influence plant growth and development by modulating processes from germination and cell elongation to senescence.16,41 Maintenance of a high level of antioxidant capacity is an important mechanism by which maize seedlings overcome the negative physiological effects resulting from exposure to low R:FR reflected from neighbouring weeds. Thiamethoxam is known to
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Thiamethoxam alters maize seedlings response to weeds induce antioxidant activity within treated plants by increasing the radical-scavenging antioxidant activity of DPPH measured within the phenolic extract of maize seedling leaves2 or by enhancing ascorbic and salicylic acid levels.9,10 Thus, the maintenance of high antioxidant activity by thiamethoxam would account for the observed enhancement in seedling vigour, reported previously.5,6 Low R:FR reflected from neighbouring weeds has been reported previously to modulate the phenylpropanoid pathway, resulting in a reduction in the anthocyanin content and an enhancement in lignin synthesis in maize seedlings.24 In this study, thiamethoxam was able to maintain the expression of CHS and CAD and consequently maintain the anthocyanin and lignin levels even under weedy conditions. This finding is supported by the fact that thiamethoxam was reported recently to enhance the phenyl ammonia layase (PAL) activity, a key enzyme in the phenylpropoanoid pathway, the site of anthocyanin and lignin biosynthesis.5,7
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CONCLUSIONS
Under non-limiting resource conditions, thiamethoxam was found to enhance seedling vigour and to overcome the expression of typical shade avoidance characteristics caused by neighbouring weeds. The physiological mechanisms by which these results were achieved were attributed to an enhancement in seedling vegetative growth, the maintenance of the phenolics content and the activation of scavenging genes, which reduced the accumulation of H2 O2 in plant tissues. This reduction in H2 O2 content within plant tissues reduced the extent to which cellular damage occurred. This would reduce the energy expended for cellular repair and allow for this energy to be allocated to growth and maintenance of plant tissues. In addition, this work has several implications for the role of seed treatments in agriculture. Normally, seed treatments are thought of only in terms of insect and disease control. The results of the present study, however, suggest the possibility of exploring entirely new chemistries and new modes of action to enhance free radical scavenging and activate genes involved in the antioxidant defence system. This new role for seed treatments may be critical in the development of crop hybrids and cultivars that are more stress tolerant to weed competition.
ACKNOWLEDGEMENTS The financial support of the Natural Science and Engineering Research Council of Canada, File CRDPJ 425128-11, Syngenta Crop Protection and the Ontario Ministry of Agriculture, Food and Rural Affairs is gratefully acknowledged. The authors would also like to express their appreciation to Harold Wright from Syngenta and to Dr Steven Rothstein from the Department of Molecular and Cellular Biology, University of Guelph, for their contributions throughout this study.
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Pest Manag Sci 2015; 71: 505–514
