Glucosinolate Accumulation and Related Gene Expression in Pak

Article pubs.acs.org/JAFC

Glucosinolate Accumulation and Related Gene Expression in Pak Choi (Brassica rapa L. ssp. chinensis var. communis [N. Tsen & S.H. Lee] Hanelt) in Response to Insecticide Application Biao Zhu,† Jing Yang,† Yong He, Yunxiang Zang, and Zhujun Zhu* The Key Laboratory for Quality Improvement of Agricultural Products of Zhejiang Province, College of Agricultural and Food Science, Zhejiang A&F University, Hangzhou 311300, China ABSTRACT: Glucosinolates and their breakdown products are well-known for their cancer-chemoprotective functions and biocidal activities against pathogens and generalist herbivores. Insecticides are commonly used in the production of pak choi (Brassica rapa L. ssp. chinensis var. communis [N. Tsen & S.H. Lee] Hanelt). We studied the effects of four commonly used insecticides, namely, β-cypermethrin, acephate, pymetrozine, and imidacloprid, on glucosinolate metabolism in pak choi. All insecticides significantly increased both the transcription of glucosinolate biosynthetic genes and the aliphatic and total glucosinolate accumulations in pak choi. β-Cypermethrin and acephate caused gradual and continuous up-regulation of gene expression from 0.5 to 24 h after treatment, whereas pymetrozine and imidacloprid did so more rapidly, reaching a peak at 1 h and returning to normal at 3 h. Our findings indicate that the four insecticides affect glucosinolate metabolism in pak choi plants to various degrees and suggest that glucosinolates may be involved in plant insecticide metabolism. KEYWORDS: insecticide, glucosinolate, pak choi, pymetrozine, imidacloprid, β-cypermethrin, acephate

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regulation.13 In plants, the glucosinolate system acts as an efficient defense against environmental stresses such as predation by herbivores, microorganisms, fungi, wounding, and chemical poisoning.14,15 The defense and detoxification functions of glucosinolates and their breakdown products have been widely studied and demonstrated in Arabidopsis. Bednarek et al. have reported that glucosinolates are recruited for broadspectrum antifungal defense responses in living plants.16 Clay et al. found that glucosinolate metabolites are required for the Arabidopsis innate immune response.17 Previous studies by Brader have shown that aromatic glucosinolate metabolites mimic a signaling molecule in the salicylic acid-mediated defense systema component of the known systemic acquired resistance (SAR) pathway.18,19 Aliphatic glucosinolates that exist in high concentrations in pak choi and other Brassicaceae species are important for their resistance to pests.20 Although plant glucosinolate metabolism has been well studied during the past decades,8,13−19 little is known about the effects of insecticides on glucosinolate concentrations and profiles. In this study, we therefore investigated the effects of four insecticides from three major classes, neonicotinoids, pyrethroids, and organophosphorus compounds, on glucosinolate accumulation and related gene expression in two pak choi cultivars.

INTRODUCTION The use of insecticides is an effective strategy for controlling pests and is beneficial for high crop yields. Although current food supply levels are difficult to imagine without the use of synthetic insecticides, their excessive or improper application can cause substantial adverse effects, such as insecticide residues and crop losses, resulting in potential risks to human health.1 Pesticides, comprising fungicides, herbicides, and insecticides, have been shown to affect various aspects of plant life, such as plant growth, reproductive organ development, nitrogen and carbon metabolism, photosynthetic rate, antioxidant enzyme activity, nutritional components, and phytoalexins.2−5 Several studies have demonstrated that insecticides directly or indirectly influence the secondary metabolism of products such as flavonoid and phenolic compounds through nonspecific mechanisms.6,7 Nevertheless, the effect of insecticides on the metabolism of glucosinolates, important secondary metabolites of cruciferous plants, has rarely been investigated. Glucosinolates are the major class of secondary metabolites found in Brassicaceae species.8 On the basis of the various structures of their side chains and divergent hydrolyzing pathways, glucosinolates and their breakdown products have multiple biological activities in both human health and plant defense systems.9 Numerous studies indicate that glucosinolates such as glucoraphanin and glucobrassicin and glucosinolate breakdown products such as isothiocyanates can induce carcinogen inactivation through inhibition of phase I enzymes and induction of phase II enzymes; therefore, the consumption of glucosinolate-rich vegetables is in discussion to reduce the risk of cancer formation.10−12 Glucosinolates play an essential role in plant growth and development. Bone and Rossiter have indicated that glucosinolate degradation products are involved in defense against pests and phytopathogens and potentially participate in nitrogen and sulfur metabolism and growth © XXXX American Chemical Society

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MATERIALS AND METHODS

Plant Materials. Seeds of pak choi (Brassica rapa L. ssp. chinensis var. communis [N. Tsen & S.H. Lee] Hanelt) cultivar Hangzhou You Dong Er (HZYDE) and Si Yue Man (SYM) were germinated and Received: August 8, 2015 Revised: October 12, 2015 Accepted: October 20, 2015

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DOI: 10.1021/acs.jafc.5b03894 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Table 1. Effects of Different Insecticide Treatments on Fresh Weight, Dry Weight, and Dry Matter Contents of Shoots of Pak Choia cultivar

treatment

CK

PYT

PCL

GXLQJZ

YXJAL

HZYDE

fresh wt (g/pot) dry wt (g/pot) dry matter content (%)

37 ± 1.32 c 2.64 ± 0.15 bc 7.14 ± 0.46 a

40.7 ± 1.04 ab 2.86 ± 0.09 ab 7.04 ± 0.19 a

41.8 ± 1.44 a 3.20 ± 0.28 a 7.65 ± 0.57 a

32.2 ± 1.53 d 2.26 ± 0.10 c 7.04 ± 0.35 a

38.5 ± 1.73 bc 2.84 ± 0.23 ab 7.36 ± 0.32 a

SYM

fresh wt (g/pot) dry wt (g/pot) dry matter content (%)

37 ± 1.32 b 2.64 ± 0.15 cd 7.14 ± 0.46 a

46.2 ± 2.08 a 3.05 ± 0.18 ab 6.62 ± 0.19 a

47.7 ± 0.58 a 3.37 ± 0.21 a 7.07 ± 0.50 a

38.3 ± 0.76 b 2.53 ± 0.23 d 6.60 ± 0.73 a

45 ± 1.00 a 2.92 ± 0.16 bc 6.50 ± 0.35 a

CK, control; PYT, pymetrozine; PCL, imidacloprid; GXLQJZ, β-cypermethrin; YXJAL, acephate. Data are the mean ± standard deviation (n = 3). Means followed by different letters in each row are significantly different (P < 0.05). a

designed as described previously.23 Gene expression was normalized using Brassica actin (EX087730) as the housekeeping gene. Real-time PCR reactions were performed in triplicate on an ABI 7300 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) with the SYBR Green qPCR kit (Takara, Japan). The qRT-PCR protocol was as follows: initiated by 30 s at 95 °C, then followed by 40 cycles of 95 °C for 5 s and 60 °C for 31 s, and completed with a melting curve analysis program. All samples were run in triplicate, and the expression values obtained were normalized against actin. Analysis of the relative gene expression data was done using the 2−△△Ct method.24 Statistical Analysis. The data of plant growth and glucosinolate concentrations were analyzed using analysis of variance (ANOVA). Mean values were compared using the least significant difference (LSD) at the 0.05 significance level.

grown in vermiculite for 3 weeks, and the seedlings with three to four true leaves were transplanted to a 10 L plastic container containing aerated full-nutrient solution, and the solution was changed every 3 days.21 A randomized complete block (RCB) with three replicates was applied. All of the experiments were carried out from April 2009 to May 2009 in a greenhouse with the natural photoperiod and at an average temperature of 25 °C during the day and 10 °C at night. The highest light intensity was 650 μmol m−2 s−1. The insect screen was used to keep the plants in the absence of pests. Insecticide Treatment. After having been cultivated for 1 week, the plants were treated with the following commercial formulations of insecticides at recommended concentrations: pymetrozine (25% active ingredient, 0.5 g/L), imidacloprid (70% active ingredient, 0.7 g/L), βcypermethrin (4.5% active ingredient, 0.6 g/L), and acephate (30% active ingredient, 2.0 g/L). Both sides of pak choi leaves were sprayed until drips ran off (approximately 5 mL per plant). Spraying was performed in a spray application box with a thin layer chromatography (TLC) jet connected to a hand pump. Plants adjacent to the treated one were protected with aluminum foil. Sample Collection. Plants were harvested at 2 weeks after treatment for glucosinolate analysis. The shoots were weighed and immediately frozen in liquid nitrogen and freeze-dried. After being weighed, the samples were ground into fine powder and stored at −20 °C for glucosinolate analysis. The leaf samples of HZYDE were collected separately at 0.5, 1, 3, and 24 h after treatment and being frozen in liquid nitrogen to determine the gene expression level. Every treatment consists of three replicates with three plants per replicate. Glucosinolate Analysis. The glucosinolate extraction and analysis procedures were performed as previously described by Krumbein with slight modification.21,22 First, 0.25 g of sample powder was boiled with 10 mL of 70% methanol to inactivate myrosinase, then the supernatant was loaded onto a 1 mL mini-column (J. T. Baker, Phillipsburg, NJ, USA) to desulfate overnight with 200 μL of aryl sulfatase (SigmaAldrich Co., St. Louis, MO, USA). The mini-columns were pretreated and contained 500 μL of activated DEAE Sephadex A25 (Amersham Biosciences, Uppsala, Sweden). The resultant desulfo (ds) glucosinolates were eluted with ultrapure water and stored at −20 °C until further analysis. Samples were analyzed by high-performance liquid chromatography (HPLC) in an Agilent 1200 HPLC system (G1311A quaternary pump, G1322A vacuum solvent delivery degasser, G1316A thermostated column compartment, Agilent Technologies, Inc., Palo Alto, CA, USA) consisting of a G1329A autoinjector, a prontosil ODS2 column (250 × 4 μm, 5 μm, Bischoff, Leonberg, Germany), and a G1315B diode array detector (DAD) set at 229 nm. The mobile phase was ultrapure water (A) and acetonitrile (Tedia, Fairfield, OH, USA) (B) in a linear gradient from 0 to 20% B for 32 min, and then constant 20% B for 6 min, followed by 100% B and 0% B prior to the injection of the next sample. The flow rate was 1.3 mL min−1. Gene Expression. The RNA extraction and RT-PCR procedure were performed according to our previously described protocols.23 Briefly, total RNA was extracted from samples using TRIzol reagent RNAisoTM Plus (Takara, Japan) following the manufacturer’s protocols. Transcription levels were analyzed by quantitative realtime PCR (qRT-PCR), and the gene-specific primer sets were

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RESULTS AND DISCUSSION Plant Growth. We examined the effects of different applied insecticides on plant growth (Table 1). Pymetrozine and imidacloprid significantly increased shoot fresh and dry weights of the two cultivars compared with the control (P < 0.05). This observation is consistent with previous reports that neonicotinoid insecticides, especially imidacloprid, enhance plant growth and abiotic/biotic stress tolerance independent of their insecticidal functions.5,25 Gonias has reported that imidacloprid improves cotton crop growth and yield, with the growthenhancing effect attributed to increased chlorophyll fluorescence yield and photosynthetic rate.25 Navreet has further shown that imidacloprid improves cotton metabolism, as reflected by increased levels of Bacillus thuringiensis protein, peroxidase enzyme activity, total phenols, height, number of retained bolls on plants, and yield.5 We therefore postulate that the stimulating effect of imidacloprid on plant growth (Table 1) may also be related to its stimulatory effects on photosynthesis. The remarkable growth-enhancing effect of imidacloprid on plants in addition to its insecticidal properties has also been noted by other researchers and farmers.26,27 A yield-increasing effect was also observed in the current study in plants treated with pymetrozine, another neonicotinoid insecticide. In contrast, β-cypermethrin treatment decreased yields, and acephate had no effect (Table 1). The different effects of these insecticides on pak choi yield may provide valuable information allowing producers to select the appropriate insecticidesnot only according to their pest-killing efficiency but also based on their potential effects on plant growth. Concentrations and Profiles of Glucosinolates. The HPLC profile of DS-GSs in pak choi is shown in Figure 1. On the basis of the structures of their side chains, glucosinolates are divided into three groups: aliphatic, aromatic, and indolyl glucosinolates.8 Concentrations of total glucosinolates as well as the three individual groups are compared in Figures 2 and 3 for B


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(Figures 2 and 3). The increase in total glucosinolates due to insecticide treatment can be attributed to an increase in aliphatic glucosinolates, as the concentrations of aliphatic glucosinolates were respectively enhanced by 51.6, 56.5, 58.5, and 96.2% in SYM and by 65.5, 105.7, 21.0, and 38.3% in HZYDE (Figures 2 and 3). Indolyl and aromatic glucosinolate concentrations were also increased by insecticide treatments. In contrast to aliphatic glucosinolates, the effects of insecticides on indolyl and aromatic glucosinolates were not as obvious or consistent, with variations existing between both treatments and cultivars. For example, imidacloprid treatment significantly increased indolyl and aromatic glucosinolate levels in HZYDE by 64.8 and 87.2%, respectively (P < 0.05; Figure 2), but had no effects on SYM (Figure 3). Pymetrozine treatment significantly increased indolyl glucosinolates (by 74.3% in SYM and 83.8% in HZYDE), whereas β-cypermethrin significantly increased aromatic glucosinolates (by 111.9% in SYM and 69.6% in HZYDE) in the two cultivars (Figures 2 and 3; P < 0.05). Acephate also increased levels of indolyl and aromatic glucosinolates in the two cultivars, but the increase was not significant (Figures 2 and 3). The variation in induced glucosinolate profiles between treatments may be attributed to the different physicochemical properties of the insecticides, such as their different action modes. It was reported that pymetrozine can taken up and translocated quickly through the vascular tissue, whereas acephate has a comparably longer effect on plant by metabolization to active intermediates such as

Figure 1. HPLC chromatogram of glucosinolates isolated from pak choi. Peak numbers refer to the glucosinolates listed in Table 2. Peaks: 1, sinigrin (internal standard); 2, glucoalyssin; 3, gluconapin; 4, glucobrassicanapin; 5, glucobrassicin; 6, gluconasturtiin; 7, 4methoxyglucobrassicin; 8, neoglucobrassicin.

pak choi plants treated with different insecticides. All insecticide treatments significantly increased concentrations of total and aliphatic glucosinolates in the two cultivars (P < 0.05) compared with the control. Compared with the control, pymetrozine, imidacloprid, β-cypermethrin, and acephate treatments increased total glucosinolate concentrations by 52.8, 49.5, 56.2, and 88.4%, respectively, in the SYM cultivar and by 63.2, 101.8, 29.9, and 37.6% in the HZYDE cultivar

Figure 2. Effects of different insecticide treatments on aliphatic, indolyl, aromatic, and total glucosinolate concentrations in shoots of pak choi cultivar Hangzhou You Dong Er (HZYDE). CK, control; PYT, pymetrozine; PCL, imidacloprid; GXLQJZ, β-cypermethrin; YXJAL, acephate. Data are the mean ± standard deviation (n = 3). DW, dry weight. Means assigned different letters are significantly different in each treatment (P < 0.05). C


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Figure 3. Effects of different insecticide treatments on aliphatic, indolyl, aromatic, and total glucosinolate concentrations in shoots of pak choi cultivar Si Yue Man (SYM). CK, control; PYT, pymetrozine; PCL, imidacloprid; GXLQJZ, β-cypermethrin; YXJAL, acephate. Data are the mean ± standard deviation (n = 3). DW, dry weight. Means assigned different letters are significantly different in each treatment (P < 0.05).

imidacloprid (by 87.1%) and β-cypermethrin (by 69.6%) in the HZYDE cultivar (P < 0.05) and was increased by 39.4 and 111.9% by pymetrozine and β-cypermethrin, respectively, in the SYM cultivar (P < 0.05). The other insecticides had no notable effects. With respect to individual indolyl glucosinolates, all insecticides induced significant increases in glucobrassicin concentrations in HZYDE (P < 0.05) and slight increases in SYM (Table 2). Concentrations of 4-methoxyglucobrassicin increased under all four insecticide treatments in the two cultivars, although only the change induced by the pymetrozine treatment in SYM was significant (P < 0.05). All insecticides increased neoglucobrassicin concentrations in HZYDE (P < 0.05), whereas the effects in SYM were very diverse. In particular, treatment with pymetrozine and acephate increased neoglucobrassicin concentrations (P < 0.05), whereas imidacloprid and β-cypermethrin decreased them (Table 2). The large variation observed in the effect of insecticide treatments on individual aromatic and indolyl glucosinolates in the two cultivars is consistent with the results of previous studies in which the content of indolyl glucosinolates was greatly affected by the environment and genotype−environment interactions.35,36 The increased glucosinolate accumulation induced by insecticides in this study is hypothesized to be supported by former researchers’ finding that insecticides increase amino acid and protein levels in pak choi,4 because glucosinolates are nitrogen-based defenses derived from amino acids. Expression of Glucosinolate Biosynthetic Genes. Glucosinolate biosynthesis and accumulation are regulated by several biosynthetic genes. BrCYP83A1 and BrCYP83B1 are

methamidophos.28,29 The variation in insecticide-induced glucosinolate profiles between cultivars may simply reflect their different genetic backgrounds, as their glucosinolate contents varied markedly (Figures 2 and 3, CK). Lankau and Kliebenstein have reported that different genotypes under the same environmental stress accumulate defense metabolites differently.30,31 Aliphatic glucosinolates, which are important nutrient compounds and plant defense substrates in Brassica vegetables, play a vital role in plant resistance to pests and other stresses.20 Plants are hypothesized to use environmental cues to adjust their defense levels to minimize potential costs.32 Consequently, our observation that aliphatic and total glucosinolates increased after insecticide treatments supports the idea that insecticides can change secondary metabolite contents in plants; this is especially true for glucosinolates, as they are important defense compounds involved in responses to diverse environmental stresses in Brassicaceae species. Individual glucosinolates were also influenced by insecticides and exhibited greater variation, both between treatments and between cultivars (Table 2). Individual aliphatic glucosinolates, including glucoalyssin, gluconapin, and glucobrassicanapin, all significantly increased after treatments of the four insecticides, thus leading to a significant increase in aliphatic glucosinolates compared with the control (see details in Table 2; P < 0.05). The similar response patterns of the individual aliphatic glucosinolates can be attributed to their interactive biosynthetic pathways, as the three glucosinolates can be transferred through the same regulatory network.33,34 The level of gluconasturtiin, the only aromatic glucosinolate, was significantly increased by D


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two key synthetic genes involved in the conversion of aldoximes to thiohydroximic acids in the glucosinolate biosynthesis pathway of pak choi. BrCYP83A1 mainly metabolizes aliphatic oximes, whereas BrCYP83B1 is mostly responsible for aromatic and indolic oximes. BrCYP83A1 and BrCYP83B1 expression levels were examined by qRT-PCR. Consistent with increased glucosinolate accumulation, BrCYP83A1 and BrCYP83B1 were significantly overexpressed in response to the insecticide treatments (Figure 4). Peak

a CK, control; PYT, pymetrozine; PCL, imidacloprid; GXLQJZ, β-cypermethrin; YXJAL, acephate. Data are the mean ± standard deviation (n = 3). DW, dry weight. Means followed by different letters in each row are significantly different (P < 0.05).

e bcd e a cde 0.021 0.101 0.032 0.110 0.023 ± ± ± ± ± 0.287 0.400 0.253 0.608 0.344 bc a bc c a 0.006 0.035 0.035 0.007 0.089 ± ± ± ± ± 0.222 0.339 0.213 0.157 0.325 b a b b b 0.025 0.009 0.011 0.015 0.027 ± ± ± ± ± 0.086 0.185 0.089 0.098 0.104 0.029 c 0. 022 a 0.050 c 0.028 bc 0.053 bc ± ± ± ± ± 0.194 0.351 0.208 0.232 0.253 e c b a de 0.078 0.012 0.073 0.105 0.013 ± ± ± ± ± 0.400 0.704 1.062 1.349 0.487 0.836 1.100 0.783 0.525 1.190 ± ± ± ± ± 0.007 0.010 0.044 0.025 0.004 ± ± ± ± ± CK PYT PCL GXLQJZ YXJAL SYM

0.056 0.119 0.214 0.183 0.071

e c a ab de

5.467 8.158 8.049 7.856 11.064

de b b bc a

e e b bc de 0.047 0.059 0.044 0.016 0.046 ± ± ± ± ±

gluconasturtiin

0.248 0.254 0.464 0.420 0.292 c a ab a bc 0.032 0.050 0.059 0.047 0.058 ± ± ± ± ±

neoglucobrassicin

0.162 0.314 0.254 0.321 0.214 c c c c c 0.003 0.011 0.010 0.015 0.013 ± ± ± ± ±

4-methoxyglucobrassicin

0.024 0.049 0.045 0.045 0.045 d bc bc b c 0.030 0.028 0.036 0.059 0.015 ± ± ± ± ±

glucobrassicin

0.126 0.210 0.213 0.281 0.192 e c b e cd 0.061 0.059 0.193 0.061 0.032 ± ± ± ± ± 0.447 0.758 1.094 0.356 0.621

glucobrassicanapin

f de cd ef ef 0.329 0.594 0.606 0.364 0.463 ± ± ± ± ±

gluconapin

3.322 5.459 6.651 4.208 4.553 cde ab ab cd b 0.008 0.031 0.021 0.017 0.028 CK PYT PCL GXLQJZ YXJAL

glucoalyssin treatment cultivar

HZYDE

± ± ± ± ±

indolyl GS aliphatic GS

Table 2. Effects of Different Insecticide Treatments on the Concentrations of Individual Glucosinolates (GS) in Shoots of Pak Choia

0.092 0.172 0.198 0.110 0.166

aromatic GS


Figure 4. Effect of insecticide treatments on expression of the glucosinolate biosynthetic gene BrCYP83A1 (white) and BrCYP83B1 (black) in leaves of pak choi cultivar Hangzhou You Dong Er (HZYDE). CK, control; PYT, pymetrozine; PCL, imidacloprid; GXLQJZ, β-cypermethrin; YXJAL, acephate. Time = 0 h for each treatment was set as 1; the error bar on each column corresponds to the standard deviation from three biological replicates.

insecticide-induced expression levels of the two genes were 18 (pymetrozine), 3 (imidacloprid), 4 (β-cypermethrin), and 4 (acephate) times higher compared with the control in the case of BrCYP83A1, and 10 (pymetrozine), 3 (imidacloprid), 3 (βcypermethrin), and 5 (acephate) times higher for BrCYP83B1. These results indicate that the induction effects of pymetrozine were much higher than those of the other three insecticides (Figure 4). In addition to maximum expression levels, the patterns of expression over time varied among different E


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activities against environmental stresses. We conclude that insecticides protecting cruciferous plants from pests act not only through their direct biocidal effects on insects but also by indirectly enhancing plant defense capacity. Additionally, the different expression patterns of glucosinolate biosynthetic genes induced by insecticides suggest that insecticides possess different actions that affect glucosinolate metabolism. On this last point, however, further detailed investigation is needed.

insecticides, with pymetrozine and imidacloprid following one trend and β-cypermethrin and acephate another. Pymetrozine and imidacloprid rapidly induced BrCYP83A1 and BrCYP83B1 expression, which respectively peaked at 0.5 and 1 h after insecticide treatment and then decreased to normal levels at 3 h. Interestingly, the two genes induced by β-cypermethrin and acephate treatment were expressed gradually after insecticide treatment, reaching a peak at 3 h after treatment and remaining up-regulated until 24 h. With respect to their different physicochemical properties, insecticides have different modes of action toward pests as well as plants. Among the four studied insecticides, pymetrozine is a neuroactive systemic insecticide with a remarkable selectivity for plant-sucking pests, whereas imidacloprid is a systemic insecticide active against numerous sucking and biting insects. β-Cypermethrin is a systemic broad-spectrum insecticide that acts as a contact and stomach poison against sucking and biting pests, and acephate is a very effective systemic organophosphorus insecticide functioning as both a contact and a stomach poison.28,37−41 Neuroactive systemic insecticides work more rapidly than stomach insecticides.42 Interestingly, our study results also show that the two neonicotinoid insecticides, pymetrozine and imidacloprid, up-regulated the expression of two glucosinolate biosynthetic genes more rapidly than did βcypermethrin and acephate, the two stomach poison pesticides. Phloem mobility is an important determinant of the effectiveness of an insecticide in a plant. All of the examined insecticides can be absorbed by leaf tissue immediately after application, but their mobility patterns differ within plants.28,39,43−45 Pymetrozine has the best phloem mobility, as evidenced by a study in which radioactive 14C-labeled pymetrozine was able to reach sugar beet leaf petioles, especially those of young leaves, within 4 h.28 The higher phloem mobility of pymetrozine may therefore explain its faster up-regulation of the two genes involved in glucosinolate synthesis (Figure 4). In addition to phloem mobility, the halflife of these insecticides in plants may be another important factor influencing the duration of induced expression of genes related to glucosinolate accumulation. The duration of insecticide effectiveness in plants depends on their half-lives, which largely determine their phytotoxic effect on plant metabolism.46 Previous investigations have revealed that the half-lives of these insecticides are 1.65 days in tomato for pymetrozine, 2−4 days in cabbage for imidacloprid, 3.7 days in cabbage for β-cypermethrin, and about 3.1−13.5 days in most crops for acephate.47−50 The durations of insecticide-induced glucosinolate gene expression were fairly consistent with the respective half-lives of the insecticides. As shown in Figure 4, pymetrozine and imidacloprid treatments had rapid shortduration effects, whereas β-cypermethrin and acephate acted more slowly for a longer duration. We therefore conclude that the different temporal gene expression patterns induced by various insecticides were correlated with their different action modes and their varying physicochemical properties, such as phloem mobility and half-life in plants. Over the past several decades, our understanding of the molecular mechanisms by which plants respond and adapt to biotic stresses has drastically improved. The present study suggests that the glucosinolate−myrosinase system plays an important part in plant response to insecticides. The significantly increased glucosinolate accumulation induced by insecticides is beneficial, both in regard to the nutritional value of pak choi for human health and in plant biological defense

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AUTHOR INFORMATION

Corresponding Author

*(Z.Z.) Mail: The Key Laboratory for Quality Improvement of Agricultural Products of Zhejiang Province, School of Agricultural and Food Science, Zhejiang A&F University, Huan Cheng Bei Lu 88, Lin’an, Hangzhou 311300, China. Phone: +86 571 63743001. Fax: +86 571 63741276. E-mail: [email protected]. Author Contributions †

B.Z. and J.Y. contributed equally to this work.

Funding

This research was financially supported by the National Natural Science Foundation of China (31501748, 31201620, 31572130, 31572115), Zhejiang Provincial Natural Science Foundation of China (LZ14C150001), and the Development Fund of Zhejiang A&F University (2013FR005). Notes

The authors declare no competing financial interest.

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REFERENCES

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Glucosinolate Accumulation and Related Gene Expression in Pak

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