Differences in Root Physiological and Proteomic Responses to Dibutyl

Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

pubs.acs.org/JAFC

Differences in Root Physiological and Proteomic Responses to Dibutyl Phthalate Exposure between Low- and High-DBPAccumulation Cultivars of Brassica parachinensis Hai-Ming Zhao,†,‡,§ He-Biao Huang,†,§ Yu-Mei Luo,† Chun-Qing Huang,† Huan Du,† Lei Xiang,† Quan-Ying Cai,† Yan-Wen Li,† Hui Li,† Ce-Hui Mo,*,† and Zhenli He‡

J. Agric. Food Chem. Downloaded from pubs.acs.org by UNIV OF WINNIPEG on 12/14/18. For personal use only.

†

Guangdong Provincial Research Center for Environment Pollution Control and Remediation Materials, College of Life Science and Technology, Jinan University, Guangzhou 510632, China ‡ Indian River Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Fort Pierce, Florida 34945, United States S Supporting Information *

ABSTRACT: Di-n-butyl phthalate (DBP), as an endocrine-disrupting chemical that tends to be accumulated in crops, poses great risks to human health through the food chain. To identify the molecular mechanism underlying differences in their DBP accumulation, the root physiological and proteomic responses to DBP stress of two Brassica parachinensis cultivars, a high-DBP accumulator (Huaguan) and a low-DBP accumulator (Lvbao), were investigated. Root damage of greater severity and significantly greater (p < 0.05) decreases in root protein content and root activity were detected in Lvbao than in Huaguan, suggesting that Lvbao had lower tolerance to DBP. In total, 52 DBP-responsive proteins were identified by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. More proteins involved in basic metabolic processes, such as protein synthesis and energy metabolism, were downregulated in Lvbao, possibly explaining its lower tolerance and root damage. Several proteins involved in starch metabolism, cell-wall biosynthesis and modification, and stress response were activated in Huaguan, suggesting greater tolerance to DBP. Overall, differences in root proteome between the two cultivars might be responsible for the genotype-dependent DBP tolerance and accumulation in B. parachinensis. KEYWORDS: Brassica parachinensis cultivar, dibutyl phthalate (DBP), accumulation variation, proteome analysis, molecular basic

1. INTRODUCTION The widespread use of phthalate-containing products has increased the frequency of phthalate ester (PAE) contamination of agricultural environments.1 Agricultural soils and vegetables have been polluted by PAEs; indeed, the PAE concentrations in most agricultural soils in China are in the milligram-per-kilogram range.2,3 Dibutyl phthalate (DBP), one of the most abundant PAEs, is listed as a priority pollutant by China National Environmental Monitoring and by the US Environmental Protection Agency. DBP in soils can be taken up and accumulated by crops, especially leafy vegetables, and represents a threat to human health via the food chain.1,4,5 Therefore, strategies to reduce the DBP content in vegetables are needed, e.g., breeding of crop cultivars with safe levels of pollutants in the edible parts.6 Brassica parachinensis is a leafy vegetable native to southern China and is exported globally. B. parachinensis tends to accumulate DBP when growing in DBP-contaminated soils.7 The Huaguan genotype of B. parachinensis shows marked DBP uptake and accumulation and the Lvbao genotype relatively little.5 When grown in DBP-contaminated soil [10 mg/kg dry weight (DW)], the shoot DBP concentrations differed by 3.7fold between the two cultivars. The low DBP concentration in Lvbao shoots was mainly attributable to relatively inefficient DBP uptake and translocation by roots.5 Furthermore, genotype-dependent pollutant (including DBP) accumulation is dependent on root bioprocesses.5,8,9 Therefore, information © XXXX American Chemical Society

on these root bioprocesses would assist elucidation of the mechanism underlying low DBP accumulation and enable establishment of effective crop breeding programs. However, few studies have focused on the molecular mechanisms of organic pollutant uptake and accumulation in plants,10 although several reports on heavy metals have been published.11 Proteomic approaches can be useful to understand the underling mechanisms used by plants to cope with DBP-toxicity on a whole-plant basis. However, to date little proteomic information is available about crop responses to the DBP stress. Two-dimensional electrophoresis (2-DE) has been the dominant platform in quantitative plant proteomics for a long time because it is a very useful, cheap, and convenient method for studying different aspects of the plant biology at the protein level, including growth, developmental processes, and responses to environmental factors, among others.12,13 It will continue being a key technique in plant proteomics and plant biology research, especially because the resolution and sensitivity it offers are exquisite and unsurpassed if one wants a global view of cellular activity.14 The objective of this study was to identify the root physiological and molecular mechanisms responsible for the Received: Revised: Accepted: Published: A

September 12, 2018 November 26, 2018 December 10, 2018 December 10, 2018 DOI: 10.1021/acs.jafc.8b04956 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

temperature and stored at −80 °C. The Bradford method was used to quantitate protein with BSA as the standard.16 2.5. Two-Dimensional Gel Electrophoresis. Purified total proteins were dissolved in rehydration buffer {5 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate, 1% DTT, 1% immobilized protein gradient (IPG) buffer (pH 4−7), and 0.01% bromophenol blue}. The solution (460 μL/gel) was loaded onto an Immobilon 24 cm IPG strip (pH 4−7; Bio-Rad) and isoelectrically focused using an Ettan IPGphor 3 apparatus (GE Healthcare) with the following program: (i) 1 h at 500 V (step and hold), (ii) 1 h at 1000 V (gradient), (iii) 8000 V for 3 h (gradient), and (iv) 8000 V for 5 h, 36 min (step and hold). Next, the IPG strips were incubated in equilibration buffer [6 M urea, 50 mM Tris-HCl (pH 8.8), 2% sodium dodecyl sulfate (SDS), 30% glycerol, and 1% DTT or 2.5% iodoacetamide]. The strips were transferred to the top of a 12% SDS−polyacrylamide gel covered with 0.8% lowmelting-point agarose gel in an Ettan DAT Six vertical electrophoresis system (GE Healthcare). Electrophoresis was performed at a constant current of 12 W/gel for 1 h followed by 17 W/gel for 4.5 h. Gels were stained with a combination of Coomassie Blue and silver and were imaged using a Microtek ScanMaker i800 Scanner (Microtek International). Band patterns were analyzed using ImageMaster 2D Platinum 5.0 software, and the significance of differences in protein levels was analyzed by the t-test (p < 0.05). 2.6. Mass Spectrometry. Stained 2-DE gels were washed several times with ultrapure water and protein spots of interest were excised. These spots were destained in a solution containing 200 mM NH4HCO3 in 40% acetonitrile for 20 min at 37 °C and dried in a vacuum. The dried spots were subjected to in-gel digestion with 0.5 μg of trypsin and reaction buffer consisting of 40 mM NH4HCO3 and 9% acetonitrile at 37 °C overnight. After digestion, the peptide samples were extracted in 0.1% trifluoroacetic acid and 50% acetonitrile and stored at −20 °C for analysis by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDITOF MS). An aliquot (1.5 μL) of sample was pipetted into the MALDI-TOF sample plate and air-dried. Next, 1.0 μL of the mixture was transferred to an α-cyano-4-hydroxycinnamic acid matrix, dried at room temperature, and analyzed by MALDI-TOF MS (Bruker Dalton). The ultraviolet laser was operated at a 200 Hz repetition rate and a wavelength of 355 nm. The acceleration voltage was 20 kV, and the maximum mass resolution was 1500 Da. BioTools (Bruker Dalton) software was used to filter the signal baseline peak and distinguish the signal peak. Protein identification was carried out by peptide mass fingerprinting (PMF) using Mascot software. Protein scores >61 were assumed to be significant (p < 0.05). Differential expression was defined as an at least 1.5-fold increase or 0.67-fold decrease between averaged gels. AgriGO (http://bioinfo.cau.edu.cn/agriGO/index. php) was applied for the annotation and functional classification of the identified proteins using the Gene Ontology (GO) and UniProt databases. We also conducted a cluster of orthologous groups of proteins (COG) analysis (http://www.geneontology.org). The subcellular localization of all the identified DBP-RPs was predicted by CELLO v.2.5 (http://cello.life.nctu.edu.tw/) and WoLF PSORT prediction (https://www.genscript.com/wolf-psort.html). GO enrichment analysis was implemented using agriGO.18 Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was carried out in KOBAS 2.0 (http://www.mybiosoftware.com/kobas-2-0annotation-identification-enriched-pathways-diseases.html).19 2.7. Quantitative Reverse Transcription-Polymerase Chain Reaction. Total RNA was extracted from DBP-treated and control plant roots in triplicate using RNAiso Plus (TaKaRa). First-strand cDNA was generated from the purified total RNA using a PrimeScript RT Reagent Kit. SYBR Green PCR amplification of triplicate 20 μL samples was performed on a CFX9600 real-time PCR system (BioRad) according to the manufacturer’s instructions. Gene expression was quantified and normalized to that of β-actin using the 2−ΔΔCT method. 2.8. Western Blot Analysis. To confirm the changes of the DBPRPs identified in 2-DE analysis, the four proteins, i.e., catalase,

variation in DBP accumulation among B. parachinensis cultivars. Various root physiological responses to DBP exposureincluding root biomass, protein content, root activity, morphology, and cellular ultrastructurewere compared between low (Lvbao) and high (Huaguan) DBPaccumulating cultivars of B. parachinensis. Two-dimensional electrophoresis was employed to explore the differences in the root proteomic responses to DBP stress, and functional maps of DBP-responsive proteins (DBP-RPs) in the two cultivars were generated. Our findings provide insight into the molecular mechanisms underlying the regulation of DBP tolerance and accumulation in B. parachinensis and may enable breeding of crop cultivars that accumulate less DBP.

2. MATERIALS AND METHODS 2.1. Plant Cultivation and Dibutyl Phthalate Treatment. The Huaguan and Lvbao cultivars of B. parachinensis were used in this study.5 A hydroponic experiment was carried out in a greenhouse at 25−32 °C (day) and 22−26 °C (night) under natural light. After seed germination in vermiculite for 2 weeks, uniform seedlings with four to five leaves were transplanted into aerated hydroponic solutions containing 0 (control), 25, 50, or 100 mg/L DBP. The hydroponic solutions were renewed every 2 days. After growth for 15 days, fresh roots were harvested, some of which were immediately frozen in liquid nitrogen and stored at −80 °C for protein extraction. Five roots were pooled per sample. The experiment was performed in biological triplicates. 2.2. Measurement of Root Protein Content and Root Activity. Root protein content was measured following the method of Zhang et al.,15 with minor modifications. Briefly, fresh root tissue (0.5 g) was ground into fine powder using a mortar and pestle. Then, 5 mL of 50 mM potassium phosphate buffer (pH 7.0), 1 mM ascorbic acid, 0.2 mM ethylenediamine tetraacetic acid (EDTA), and 2% (w/ v) polyvinylpyrrolidone were added to the powder. The mixture was centrifuged at 12 000g to remove precipitate (4 °C, 20 min), and the supernatant was subjected to a protein concentration assay using bovine serum albumin (BSA) as the standard.16 Root activity was determined following the triphenyl tetrazolium chloride (TTC) method.17 When added to tissue, TTC is reduced by dehydrogenases and the level of dehydrogenase activity is regarded as an index of plant root activity: root activity = amount of TTC reduction (μg) ÷ fresh root weight (g) × time (h). Briefly, 10 mL of both TTC (0.4%) and phosphate buffer was added to fresh root samples (0.5 g) and stored in the dark at 37 °C for 2 h. The reaction was stopped by adding 1 M H2SO4. The roots were ground, transferred to a tube containing ethyl acetate to a total volume of 10 mL, and the absorbance of the solution at 485 nm was measured. 2.3. Visualization of Root-Cell Ultrastructure. Root-cell ultrastructure was visualized as described previously.9 Root-tip fragments (1 mm3) were collected and fixed in 4% (v/v) glutaraldehyde (pH 7.2) at 4 °C overnight, postfixed in 1% (v/v) osmium tetroxide, and successively dehydrated in an ethanol series (30−100% v/v). After being embedded in Epon-812 resin, ultrathin sections (50−70 nm) were cut using a Leica UCT ultramicrotome (Leica Microsystems), mounted on copper grids, and stained with 4% (w/v) uranyl acetate and lead citrate. Samples were examined and photographed using a FEI-Tecnai G2 12-type transmission electron microscope (TEM) (Eindhoven, The Netherlands) at 100 kV. 2.4. Total Protein Extraction. Root samples were pulverized in liquid nitrogen. The powder was transferred to a 5 mL centrifuge tube and sonicated three times on ice using a high-intensity ultrasonic processor in lysis buffer [8 M urea, 1% Triton-100, 65 mM dithiothreitol (DTT), 2 mM EDTA, and 1% protease inhibitor cocktail, pH 8.0]. Debris was removed by centrifugation at 20 000g at 4 °C for 10 min and protein was precipitated by adding cold 15% trichloroacetic acid for 2 h at −20 °C. After centrifugation, the supernatant was discarded and the precipitate was washed three times with cold acetone. The protein pellet was air-dried at room B

DOI: 10.1021/acs.jafc.8b04956 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry glutathione S-transferase, ascorbate peroxidase, glutathione reductase, were selected for Western blot analysis. Proteins were extracted from the root samples as described above. Western blotting was performed according to the method previously reported.20 Proteins detected by immunostaining with anti-catalase antibody (AS09501), anti-glutathione S-transferase antibody (AS09479), anti-ascorbate peroxidase (AS08368), and anti-glutathione reductase antibody (AS06181) from Agrisera. The blots were visualized by enhanced chemiluminescence and scanned for the signal intensity of each band by using a Gel Doc XR system (Bio-Rad).

images, the root-cell ultrastructures of the two cultivars in the control were well-organized with clear cell walls, distinct mitochondria, and intact endoplasmic reticulum and plasma membrane (Figure 2a,b). However, DBP application exerted a marked effect on root-cell ultrastructure (Figure 2c,d). DBP induced greater damage to the root-cell ultrastructure in Lvbao, with obvious vacuolization in mitochondria and a reduced quantity of endoplasmic reticulum. Moreover, plasmolysis was observed in some cells, and the plasma membrane was blurred and, in some cases, ruptured (Figure 2d). In contrast, in Huaguan, DBP affected the root-cell ultrastructure only in terms of vacuole enlargement (Figure 2c). These results indicate that Huaguan roots are more tolerant to DBP than those of Lvbao. 3.3. Identification of Dibutyl Phthalate-Responsive Proteins (DBP-RPs). Representative 2-DE gels of the roots of the two cultivars are shown in Figure 3. The average numbers of protein spots in the control and DBP treatments were 886 and 1019 in Huaguan and 1102 and 821 in Lvbao, respectively. Of them, 142 and 160 protein spots showed significantly (p < 0.05) different intensities between Huaguan and Lvbao, of which 75/67 were upregulated/downregulated in Huaguan, and 52/108 were up/downregulated in Lvbao, respectively (Figure 3). As to protein spots altered by 25 mg/L DBP exposure, 75 and 67 spots in Huaguan were upregulated and downregulated, respectively, while 52 and 108 spots in Lvbao were upregulated and downregulated, respectively [Figure S1, Supporting Information (SI)]. Moreover, the intensities of most of the protein spots related to DBP stress tolerance were significantly upregulated by 25 mg/L DBP (DBP vs control, fold change >1.5) in Huaguan but unchanged/downregulated in Lvbao or unchanged in Huaguan but downregulated (fold change 0.05).

concentration (Figure 1a). The differences between the two cultivars (p < 0.05) were significant at 25 and 50 mg/L DBP for root protein content and 25 mg/L DBP for root activity (Figure 1b). Therefore, both root protein content and activity were significantly (p < 0.05) reduced by 25 mg/L DBP. 3.2. Root Characteristics and Ultrastructure. The root characteristics and cellular ultrastructure are shown in Figure 2. After 15 days of DBP treatment, the root length and fresh weight of the two cultivars were significantly decreased (p < 0.05, data not shown). Root growth in Huaguan was normal (Figure 2c); in contrast, the roots of Lvbao exhibited obvious damage, such as darkening, adhesion, and decomposition (Figure 2d). Moreover, the root biomass of Lvbao was significantly lower (p < 0.05) than that of Huaguan. In TEM C


Article


Figure 2. TEM of the root-cell ultrastructure and root characteristics (inset) of B. parachinensis Huaguan and Lvbao. (a and c) Huaguan treated with 0 (control) and 25 mg/kg DBP, respectively, and (b and d) Lvbao treated with 0 (control) and 25 mg/kg DBP, respectively. CW, cell wall; N, nucleus; ER, endoplasmic reticulum; M, mitochondrion; V, vacuole; P, plasma membrane.

3.4. Subcellular Localization and Functional Enrichment Analyses of the Identified DBP-RPs. The results of subcellular localization revealed that the majority of DBP-RPs in Huaguan were localized in the cytoplasm and extracellularly, whereas most of the identified DBP-RPs in Lvbao were localized in the cytoplasm, nucleus, and mitochondrion (Figure S3, SI). Notably, the number of the DBP-RPs localized in the cytoplasm of Huaguan cells was much higher than that of Lvbao cells, whereas the opposite trends were observed in the nucleus and mitochondrion. The other low abundant proteins were predicted to be localized in plasma membrane, vacuole, Golgi apparatus, peroxisome, and endoplasmic reticulum. To better understand the functions of DBP-RPs, GO and KEGG enrichment analyses were performed for each cultivar. The enrichment analyses were carried out with all quantitative proteins as the background set and the identified DBP-RPs as the target set. The top five GO and KEGG terms with the high enrichment scores were distinct between the two cultivars, respectively (Figure S4, SI). For Huaguan, the DBP-RPs were highly enriched in GO terms related to stress resistance, such as starch metabolic process, sulfur metabolic process, defense response (Figure S4a, SI), and KEGG pathway enrichment

analysis also suggested high enhancements of sulfur metabolism, phenylalanine metabolism, and glutathione metabolism pathways (Figure S4c, SI). By contrast, the majority of GO and KEGG terms were mainly enriched in basic biological process, such as amino acid metabolism and carbon metabolism (Figure S4b,d, SI). The results implied that Huaguan could operate the stress-related biological processes well enough to resist DBP toxicity, while Lvbao might suffer a disorder of basic metabolic processes, which partly accounted for their variation in DBP accumulation. 3.5. Analyses of Expression Levels. Nine DBP-RPs were randomly selected for qRT-PCR verification; the results generally mirrored those of MALDI-TOF MS, with the exceptions of ATP synthase β-subunit and dihydrolipoyl dehydrogenase (Figure S5, SI). Therefore, the effect of DBP on the levels of these proteins was likely due to induction of transcription of their encoding genes. Alternative explanations include post-transcriptional or post-translational modification and different protein and mRNA half-lives.21 Moreover, the qPCR results showed a significant correlation (p < 0.001) with the 2-DE data (r = 0.828, Figure 5), further indicating that the regulation of these DBP-RPs resulted from transcriptional D


Article


Figure 3. Representative 2-DE gels of root tissue of (a) Huaguan and (b) Lvbao. The numbers in the green boxes indicate protein spots (a2 and b2) upregulated and (a1 and b1) downregulated by DBP.

decrease in protein content could also be due to disruption of the endoplasmic reticulum. Regarding root-cell ultrastructure, the degree of mitochondrial vacuolization was greater in Lvbao (Figure 2d), in agreement with its greater decrease in root activity (Figure 1b). DBP reportedly exerted a similar effect in root cells of cucumber seedlings, likely due to cell membrane damage caused by excess peroxide and oxygen free radicals.15 In contrast, DBP treatment of Huaguan caused enlargement of vacuoles, leading to exclusion of DBP from the cytosol. Vacuolar enlargement and compartmentalization are important in the response of plant cells to pollutants, which involves toxin sequestering in vacuolar for pollutant detoxification.24 Overall, the root cells of Huaguan had greater tolerance to DBP. 4.2. Metabolic Proteins Were Downregulated in Lvbao. Generally, abiotic stress transiently suppresses de novo protein synthesis.25 The intensities of the four protein spots related to protein biosynthesis [S-adenosylmethionine synthase (SAMS), ubiquitin-60S ribosomal protein, DNA topoisomerase 6, and elongation factor 1] were markedly decreased in Lvbao by DBP. SAMS catalyzes the methylation of histones, nucleic acids, and phospholipids, which is required for plant development and resistance to environmental stresses.26 A low level of SAMS is associated with lipid accumulation and tissue injury.27 Ubiquitination plays a crucial role in the responses of plants to environmental stresses.28

induction of the corresponding genes in the roots of the two cultivars under DBP stress. Besides, the results determined by Western blot analysis showed that the relative abundance of all four DBP-RPs followed similar trends tested by proteomic analysis (Figure S6, SI). This, combined with the results of qPCR, suggested that the proteomic analysis results were reliable.

4. DISCUSSION 4.1. Tolerance to Dibutyl Phthalate Stress. Changes in root growth, metabolism, and energy content can directly affect the growth of above-ground plant parts.22 In the present study, DBP exerted a significant effect on the root growth of two B. parachinensis cultivars. The magnitude of the decrease in the root physiological parameters was greater in Lvbao than in Huaguan, indicating that the latter is more tolerant to DBP. Plant growth requires protein synthesis, and plants experiencing environmental stress activate protein catabolism.15 DBP reduced the protein contents in both cultivars (Figure 1a), and the decrease was of greater magnitude in Lvbao, suggesting that DBP inhibited protein synthesis. It was reported that plant was susceptible to senescence due to increased proteolysis and/or decreased protein synthesis induced by abiotic and biotic stresses.23 Also, a greater number of proteins were downregulated in Lvbao (Figure 3). The DBP-induced E


Article

Journal of Agricultural and Food Chemistry Table 1. DBP-RPs in Roots of B. parachinensis Huaguan (H) and Lvbao (L)

fold change (DBP vs CK) ± SD

DBP-RPs spot ID

UniProt accession

protein name

abbreva used in Figure 6

Q5DNB1

C104

P51423

C10

M4F3U5

B33/C43

D8L1X3

C23 B38/C37

M4CP76 M4CPX3

B34/C35 C30

M4E0A7 M4E9M4

B36/D22 B17

M4F721 Q9SD76

B23

O49299

B40/C47

M4F1I1

C59 B35/C57

M4CHC2 M4CFP7

B30

M4CQA6

B31 B37 B18 B48 C25

P13417 Q5DNA6 M4CBG7 Q7Y1T6 M4DFG6

chalcone synthase 3 dihydroflavonol-4-reductase pectinesterase polygalacturonase hexosyltransferase

B53/D27

M4DBR2

B40 B28/D19

M4EZY1 O22775

D15 B05

M4F988 M4D7W8

xyloglucan endotransglucosylase/ hydrolase UDP-D-xylose synthase 1 xyloglucan 6-xylosyltransferase 2 β-glucosidase 15 cellulose synthase

B20 B27

M4CFZ4 M4DA59

B50

M4E664

B29/C34 B59 B24 B65 B26 B57/D36 D51

M4DMG0 M4EZB1 M9PLU1 Q5DNA8 M4F4U7 W0TGY5 Q7XAW9

C50

Q93X74

B19 B39 B55 B11

M4DB15 M4DJ10 M4C7Y7 Q9M667

ATP synthase β-subunit isocitrate dehydrogenase (NADP) dihydrolipoyl dehydrogenase 6-phosphogluconate dehydrogenase α-amylase α-glucan phosphorylase 2, cytosolic probable phosphoglucomutase, cytoplasmic glyceraldehyde-3-phosphate dehydrogenase phosphoglycerate kinase probable bifunctional methylthioribulose-1phosphate dehydratase/ enolase-phosphatase UTP-glucose-1-phosphate uridylyltransferase

phenylalanine ammonialyase 4-coumarate: CoA ligase-like 6 peroxidase catalase superoxide dismutase glutathione reductase 1 glutathione S-transferase glutathione synthetase ascorbate peroxidase putative dehydroascorbate reductase monodehydroascorbate reductase glutamate-cysteine ligase ATP sulfurylase 2 chitinase family protein disease resistance protein RPP13

score

Protein Biosynthesis 43.18/5.67 271

S-adenosylmethionine synthase ubiquitin-60S ribosomal protein L40 DNA topoisomerase 6 subunit B elongation factor 1

C46

theor MW (kDa)/PI

coverage (%)

loca

cvb

33

cyto

Huaguan

Lvbao

L

0.719 ± 0.021

0.411 ± 0.025c

14.63/5.62

378

42

nuc

L

0.691 ± 0.024

0.653 ± 0.016

78.30/6.30

310

29

nuc

L

0.680 ± 0.043

0.622 ± 0.052

49.47/9.19 402 Energy and Metabolism 79.44/6.09 492 53.77/8.06 377

46

cyto

HL

2.339 ± 0.122

0.557 ± 0.008

52 44

mito mito

L HL

0.865 ± 0.054 2.44 ± 0.053

0.322 ± 0.016 0.451 ± 0.014

53.59/6.93 53.33/5.53

219 253

18 24

mito mito

HL L

1.551 ± 0.112 0.793 ± 0.033

0.644 ± 0.027 0.612 ± 0.054

α-amylase GP

43.05/5.35 95.16/5.79

373 173

39 30

extr cyto

HL H

3.312 ± 0.652 2.310 ± 0.231

3.851 ± 1.033 1.168 ± 0.305

PGMP

63.17/5.92

237

11

cyto

H

2.357 ± 0.403

1.492 ± 0.115

GADPH

47.55/6.76

199

24

extr

HL

2.462 ± 0.035

0.611 ± 0.023

PGK EA

42.40/6.28 57.47/6.11

338 110

29 16

cyto cyto

L HL

0.892 ± 0.05 2.598 ± 0.112

0.352 ± 0.003 0.643 ± 0.124

UGP 2

51.82/5.72

226

27

cyto

H

3.236 ± 0.431

1.310 ± 0.133

Wall Organization 43.06/6.15 102 42.87/5.54 278 64.22/7.19 438 42.31/6.60 245 61.96/8.97 102

25 31 40 22 7

ER ER extr extr GA

H H H H L

2.942 ± 0.136 2.944 ± 0.436 3.644 ± 0.611 3.691 ± 0.476 1.414 ± 0.221

1.484 ± 0.177 1.481 ± 0.147 1.481 ± 0.047 1.323 ± 0.142 0.658 ± 0.026

Cell CHS DRF pectinesterase PGC hexosyltransferase XTH

32.11/5.29

338

36

extr

HL

4.511 ± 1.453

2.457 ± 0.293

UDP-XS X 6-X

43.92/5.92 53.09/6.52

204 174

13 8

cyto GA

H HL

3.291 ± 0.462 3.521 ± 0.316

1.208 ± 0.367 1.609 ± 0.175

β-glucosidase cellulose synthase PAL 4CC

56.87/7.05 111.40/6.69

328 133

27 14

extr PM

L H

1.132 ± 0.068 2.832 ± 0.430

1.730 ± 0.086 1.446 ± 0.273

76.72/5.89 62.44/8.11

255 118

31 15

cyto pero

HL H

3.906 ± 0.489 2.065 ± 0.306

3.486 ± 0.348 1.269 ± 0.087

36.46/6.89 411 Stress Response and Defense 55.90/6.52 219 25.50/6.10 327 53.97/5.83 221 24.29/5.66 339 51.66/5.82 187 27.65/5.58 382 12.04/6.15 210

57

extr

H

3.53 ± 0.327

1.371 ± 0.213

28 35 27 46 22 38 46

cyto cyto cyto cyto cyto mito cyto

HL H H H H HL L

1.718 ± 0.074 2.677 ± 0.113 2.895 ± 0.153 4.011 ± 0.235 3.452 ± 0.256 2.072 ± 0.143 1.472 ± 0.242

0.647 ± 0.128 1.42 ± 0.152 1.229 ± 0.212 1.385 ± 0.277 1.482 ± 0.183 2.889 ± 0.201 2.882 ± 0.216

46.46/5.81

136

26

mito

L

1.323 ± 0.166

0.416 ± 0.073

57.60/6.02 48.40/6.04 30.05/8.84 97.27/6.14

241 328 358 87

19 30 36 17

cyto cyto vac cyto

H HL H H

2.984 3.872 2.977 2.213

± ± ± ±

1.486 ± 0.043 4.083 ± 0.264 1.334 ± 0.106 1.488 ± 0.441

peroxidase CAT SOD GR GST GS APX DHAR

γ-ECS ATP sulfurylase

F

0.061 0.129 0.182 0.339


Article

Journal of Agricultural and Food Chemistry Table 1. continued

fold change (DBP vs CK) ± SD

DBP-RPs spot ID

UniProt accession

protein name

B10 C96

V5YSJ5 M4F3B2

heat shock protein thaumatin-like protein

D49 B21/C33

M4DWY6 X2C3V5

B22

Q940H6

B25/D13

M4ES19

B41

O82132

B32

F1CMZ6

B43/D20

Q9CAF5

B72/C101

M4D359

B75

M4CNM5

B42/C70

M4FD78

abscisic acid receptor PYR1 ABA hypersensitive PP2C protein AHG3/PP2CA serine/threonine-protein kinase SRK2E mitogen-activated protein kinase dehydration-responsive element-binding protein 2A heat shock transcription factor A4a ABC transporter I family member 6 nonspecific lipid transfer protein copper transport protein ATX1 V-type proton ATPase subunit

abbreva used in Figure 6

theor MW (kDa)/PI

score

Stress Response and Defense 80.05/4.94 304 25.11/6.38 226 Signal Transduction and Transport PYR/PYL 17.46/5.24 301 PP2Cs 43.37/6.18 271

HSP

coverage (%)

loca

cvb

Huaguan

Lvbao

31 21

cyto nuc

H L

3.981 ± 0.162 0.691 ± 0.038

1.421 ± 0.087 0.452 ± 0.037

36 23

PM cyto

L HL

1.124 ± 0.160 2.884 ± 0.142

3.016 ± 0.773 0.417 ± 0.223

SnRK2

41.05/4.92

259

28

nuc

L

1.133 ± 0.143

1.998 ± 0.342

MAPK

43.05/5.00

248

27

nuc

HL

3.464 ± 0.547

2.788 ± 0.442

DREB2A

37.70/5.17

134

13

nuc

H

3.053 ± 0.253

1.466 ± 0.299

HSF

44.88/5.35

391

35

nuc

HL

3.355 ± 0.062

1.521 ± 0.083

36.93/6.62

82

6

PM

HL

2.201 ± 0.199

2.591 ± 0.083

19.89/6.53

375

41

PM

HL

4.357 ± 0.232

0.5965 ± 0.065

7.14/6.21

192

45

cyto

H

1.731 ± 0.082

1.443 ± 0.102

40.79/5.04

311

24

vac

HL

2.454 ± 0.012

0.296 ± 0.035

a

Abbreviations: loc = localization, cyto = cytoplasm, nuc = nucleus, mito = mitochondrion, extr = extracellular, ER = endoplasmic reticulum, GA = Golgi apparatus, PM = plasma membrane, pero = peroxisome, vac = vacuole. bAbbreviations: cv = cultivar, H = Huaguan, L = Lvbao. cThe significantly regulated proteins in Huaguan and/or Lvbao are highlighted in bold.

Figure 4. Functional classification of the DBP-RPs in the roots of B. parachinensis Huaguan and Lvbao. Proteins are classified on the basis of their main biological function according to the UniProt database.

Thus, the downregulation of ubiquitin-related proteins in Lvbao may explain its decreased tolerance to DBP. DNA topoisomerase and elongation factor 1 are indispensable for DNA damage repair and protein synthesis, respectively.29,30 Plants maintain their energy and metabolic homeostases, suggesting a close connection between energy availability and stress tolerance.31 Sufficient ATP is essential for biochemical and physiological activities in plants.32 DBP led to a significant decrease in the amount of ATP synthase subunit β protein in Lvbao, suggesting inhibition of ATP synthesis and suppression of root growth and development. This result is consistent with our previous report that the DBP-mediated decrease in the level of ATP synthase subunit β in leaf was of greater magnitude in Lvbao than in Huaguan.10 Following DBP treatment, the levels of enzymes related to carbohydrate metabolism, such as NADP-dependent isocitrate dehydrogenase, dihydrolipoyl dehydrogenase, and phosphogluconate dehydrogenase, were lower in Lvbao than in Huaguan, indicating greater disruption of energy metabolism in the

Figure 5. Correlation between qPCR and 2-DE analysis for the nine selected proteins. Each point represents a value of a fold change expression level in the DBP treatment compared with that in the nontreated control. Fold change values were log 10 transformed.

former. As the results of GO and KEGG enrichments showed in Figure S4 (SI), the DBP-RPs were mainly enriched in these basic metabolic processes instead of some stress-resistant related processes in Huaguan, indicating that DBP toxicity had induced physiological disorder of Lvbao. Overall, the DBPmediated downregulation of these metabolic proteins might result in the greater root damage and lower tolerance to DBP in Lvbao than in Huaguan. 4.3. Dibutyl Phthalate Tolerance in Huaguan Was Due to Enhanced Cell-Wall Biosynthesis and Modification. The plant cell wall is a dynamic network of cellulose G


Article


Figure 6. Major metabolic pathways involved in DBP tolerance and accumulation in root cells of B. parachinensis Huaguan and Lvbao. Yellow boxes denote enzymes; expression heat maps are shown on or near the arrows. Solid arrows between two metabolites represent the directions of reactions, and dashed arrows denote partial substitution patterns.

Pectins are responsible for the binding and immobilization of pollutants in the plant cell wall.38 Low-methyl-esterified pectins contain free carboxyl groups that function as negatively charged binding sites, which facilitates adsorption and accumulation of pollutants in the cell wall.36 Pectinesterase and polygalacturonase, which catalyze pectin demethylesterification, were significantly upregulated only in Huaguan (Figure 6), suggesting enhanced DBP adsorption. Hexosyltransferase, which is involved in pectin biosynthesis, was significantly downregulated in Lvbao.39 Eticha et al.40 reported that a decrease in pectin content reduced aluminum accumulation and resistance. Therefore, differences in the cell-wall pectin content and degree of methylation likely contributed to the genotype-dependent difference in DBP tolerance in B. parachinensis. Lignin deposition enables adjustment of cell-wall thickness and adaptive responses to environmental cues.41 Lignification involves several phenolic substrates and enzymes.42 In this study, nine of the identified proteins were related to phenylpropanoid synthesis and metabolism and participated in the biosynthesis of lignin and phenolic compounds (Figure 6). Most of these proteins were upregulated in Huaguan, but some were downregulated in Lvbao, suggesting a premature response to cell-wall damage in the former. This finding is in agreement with a previous report that the expression of genes related to lignin biosynthesis is higher in the roots of highcadmium-accumulating plants.35,43 Therefore, the high DBP accumulation and tolerance in Huaguan roots may be due to enhanced cell-wall biosynthesis and modification.

microfibrils and hemicellulose embedded in a pectin matrix and plays an important role in the growth and response to various stresses on plants.33 Over 55% of DBP applied to roots accumulates in the cell wall.5 Almost one-quarter of the DBPRPs were associated with cell-wall organization, suggesting the involvement of these proteins in the genotype-dependent DBP tolerance of B. parachinensis. For instance, the starch metabolism pathway is related to cell-wall biosynthesis;34 the levels of most of the DBP-RPs involved in this pathway were higher in Huaguan than in Lvbao (Figure 6 and Table 1). Moreover, they were also highly enriched in the three GO ontologies (Figure S4a, SI). UGP2 and cellulose synthase, which catalyze the biosynthesis of cellulose from glucose-1-phosphate, were upregulated only in Huaguan (Table 1, Figure 6). This may facilitate maintenance of cell-wall integrity and turgor pressure in Huaguan, thus allowing continuous root growth under DBP stress.35 Xyloglucan, the main component of hemicellulose, strengthens the cell wall by interacting with cellulose and/or lignin.36 The DBP-mediated upregulation of enzymes related to xyloglucan synthesis (UDP-D-xylose synthase 1 and xyloglucan 6-xylosyltransferase 2) was of greater magnitude in Huaguan than in Lvbao (Figure 6). Moreover, the upregulation of xyloglucan endotransglucosylases/hydrolases (XTHs) was significantly greater in Huaguan than in Lvbao, indicating greater cell-wall-remodeling activity in the former.37 This, together with the increased cellulose and xyloglucan contents, likely enhanced cell-wall flexibility in Huaguan, thereby increasing its DBP tolerance and accumulation. H


Article

Journal of Agricultural and Food Chemistry 4.4. Stress and Defense Responses Were More Active in Huaguan. Abiotic stresses lead to generation of excess reactive oxygen species (ROS), which is countered by the antioxidant system.44 In this study, catalase, superoxide dismutase, glutathione reductase (GR), glutathione S-transferase, and ascorbate peroxidase (APX) were found to be DBPRPs, most of which were upregulated only in Huaguan (Table 1), which may explain its higher DBP tolerance compared with Lvbao. Additionally, the DBP-mediated downregulation of catalase in Lvbao, which is in line with our previous reports,8,10 indicated reduced protection against DBP toxicity. Sulfur and glutathione (GSH) metabolism is a mechanism of xenobiotic detoxification and is a part of the antioxidant system.45 The KEGG pathway enrichment suggested an obvious enrichment of sulfur and glutathione metabolism pathways as well as GO enrichment in Huaguan (Figure S4a, SI). GSH biosynthesis from inorganic sulfate requires sulfur assimilation and cysteine biosynthesis (Figure 6). The DBPmediated upregulation of three key enzymes in this process (ATP sulfurylase, glutamate−cysteine ligase/γ-ECS, and glutathione synthetase) was of greater magnitude in Huaguan than in Lvbao. Moreover, the upregulation of GR in Huaguan resulted in a high GSH/glutathione disulfide ratio. GSH is a marker of oxidative stress and plays a multifaceted role in metabolism; a high GSH content protects plants against abiotic stresses.46 Therefore, the increased GSH level in Huaguan may be linked to its greater DBP tolerance and accumulation. In contrast, the ascorbate (ASA)−GSH cycle is involved in ROS scavenging and alleviation of membrane damage in plants exposed to various stresses.46 This cycle comprises the interdependent nonenzymatic antioxidants ASA and GSH, as well as the antioxidant enzymes APX, GR, and dehydroascorbate reductase (DHAR).47 The DBP-mediated upregulation of APX and DHAR was of greater magnitude in Lvbao than in Huaguan, resulting in a higher ASA/DHAR ratio, indicative of a different response to ROS stress in the former. Heat-shock protein, chitinase, and disease-resistance protein were upregulated only in Huaguan (Table 1), which might be related to its superior tolerance to DBP. Heat-shock proteins are responsible for protein folding, assembly, translocation and degradation in many normal cellular processes, and stabilizing proteins and membranes and can assist in protein refolding under various stress conditions.48 Herein, they might be crucial for cellular homeostasis in protecting Huaguan against DBP stress. Chitinase proteins are members in the group of the diseaseresistance proteins, which are involved in plant abiotic stress responses as noted for osmotic, salt, cold, wounding, and heavy-metal stresses, as well as pathogen stress.49 Thus, the upregulation of these proteins in Huaguan roots partly resulted in a higher resistance to DBP toxicity than that in Lvbao roots. 4.5. Differences in Signal Transduction and Cell Transport between the Two Cultivars under DBP Stress. To respond to abiotic stresses, plants have developed mechanisms to rapidly perceive environmental changes.50 Stress perception is followed by signal transduction from the cell surface to the nucleus, resulting in activation of gene transcription.51 Several of the DBP-RPs identified were involved in abscisic acid (ABA) signaling and the mitogenactivated protein kinase (MAPK) pathways (Table 1). ABA regulates many aspects of plant development in response to unfavorable environmental stresses.52 The two cultivars showed differences in activation of the PYR/PYL−PP2Cs− SnRK 2s central signaling complex of this pathway (Figure 6).

In the root of Lvbao, PYR/PYL and SnRK2E were upregulated and PP2CA was downregulated, indicating activation of ABA signaling.35 As reviewed by Kim et al.,53 upregulation of PYR/ PYL alleviates PP2C-mediated inhibition of SnRK 2s, leading to activation of the downstream transcription factor ABF. The transcriptional activator DREB2A, which is involved in ABAmediated abiotic stress tolerance, is upregulated in Lvbao.52,53 However, in Huaguan root, DBP did not influence the level of PYR/PYL or SnRK2E but upregulated that of PP2CA, suggesting a different ABA signal transduction mechanism (Figure 6). Upregulation of a MAPK in both cultivars suggested activation of the MAPK pathway, which is involved in the development and stress response of plants.50 These results are consistent with the findings of our previous leaf proteomic study.10 Overall, signal transduction played a role in the genotype-dependency of the response to DBP stress and may in part explain the difference in DBP accumulation and tolerance between Huaguan and Lvbao. Three kinds of proteins involved in cell transport, including nonspecific lipid transfer protein/nsLTP, copper transport protein, and V-type proton ATPase, were significantly upregulated in Huaguan, while unchanged or downregulated in Lvbao (Table 1). The three-dimensional structure of nsLTPs reveals an internal hydrophobic cavity that comprises the lipid binding site, resulting in a broad lipid-binding specificity in vitro, which is responsible for the shuttling of lipids between cell membranes and the intracellular transport of lipids.54 DBP as a typical lipid compound might be transported by nsLTPs in plant cells. Therefore, the upregulated nsLTP in Huaguan might promote DBP transport and accumulation in root cells. Copper transport proteins play an important role in copper and iron homeostasis in plants against the challenges of oxidative stress.55 The V-type proton ATPase is indispensable for plant growth under normal conditions due to its role in energizing secondary transport and maintenance of solute homeostasis. Under stress conditions such as salinity, drought, cold, acid stress, anoxia, and excess heavy metals in the soil, survival of the cells depends strongly on maintaining or adjusting the activity of the V-ATPase.56 Herein, the upregulation of these two kinds of proteins in Huaguan might partly account for its higher tolerance to DBP stress, while their inhibition in Lvbao could have contributed to the disruption of its root cells (Figure 2D). In conclusion, the root responses of the Huaguan and Lvbao cultivars of B. parachinensis to DBP differed significantly at the physiological and proteomic levels. Huaguan exhibited superior root protein content, activity, characteristics, and cellular ultrastructure compared to Lvbao. A greater number of identified proteins involved in energy and metabolism, cellwall biosynthesis and modification, the stress and defense responses, and signal transduction were activated in Huaguan, indicating superior maintenance of physiological homeostasis in this cultivar. Such data can be used to mine novel functional genes, identify functionally relevant molecular tags, and determine the regulatory protein networks, which can subsequently be utilized for generating pollution-safe crop varieties. We report here the molecular mechanisms underlying the intergenotype differences in DBP accumulation in B. parachinensis, which will facilitate screening for and breeding of crop cultivars that accumulate low levels of pollutants. I


Article


■

Food Safety. In Twenty Years of Research and Development on Soil Pollution and Remediation in China; Springer: Singapore, 2018; pp 413−427. (7) Mo, C. H.; Cai, Q. Y.; Tang, S. R.; Zeng, Q. Y.; Wu, Q. T. Polycyclic aromatic hydrocarbons and phthalic acid esters in vegetables from nine farms of the Pearl River Delta, South China. Arch. Environ. Contam. Toxicol. 2009, 56 (2), 181−189. (8) Zhao, H. M.; Du, H.; Xiang, L.; Li, Y. W.; Li, H.; Cai, Q. Y.; Mo, C. H.; Cao, G.; Wong, M. H. Physiological differences in response to di-n-butyl phthalate (DBP) exposure between low- and high-DBP accumulating cultivars of Chinese flowering cabbage (Brassica parachinensis L.). Environ. Pollut. 2016, 208, 840−849. (9) Zhao, H. M.; Huang, H. B.; Du, H.; Lin, J.; Xiang, L.; Li, Y. W.; Cai, Q. Y.; Li, H.; Mo, C. H.; Liu, J. S.; et al. Intraspecific variability of ciprofloxacin accumulation, tolerance, and metabolism in Chinese flowering cabbage (Brassica parachinensis). J. Hazard. Mater. 2018, 349, 252−261. (10) Zhao, H. M.; Huang, H. B.; Du, H.; Xiang, L.; Mo, C. H.; Li, Y. W.; Cai, Q. Y.; Li, H.; Liu, J. S.; Zhou, D. M.; Wong, M. H. Global picture of protein regulation in response to dibutyl phthalate (DBP) stress of two Brassica parachinensis cultivars differing in DBP accumulation. J. Agric. Food Chem. 2018, 66 (18), 4768−4779. (11) Clemens, S.; Ma, J. F. Toxic heavy metal and metalloid accumulation in crop plants and foods. Annu. Rev. Plant Biol. 2016, 67, 489−512. (12) Oliveira, B. M.; Coorssen, J. R.; Martins-de-Souza, D. 2DE: the phoenix of proteomics. J. Proteomics 2014, 104, 140−150. (13) Jorrin-Novo, J. V.; Komatsu, S.; Sanchez-Lucas, R.; Rodriguez de Francisco, L. E. Gel electrophoresis-based plant proteomics: Past, present, and future. Happy 10th anniversary Journal of Proteomics! J. Proteomics 2018, DOI: 10.1016/j.jprot.2018.08.016 (14) Naryzhny, S. Inventory of proteoforms as a current challenge of proteomics: Some technical aspects. J. Proteomics2019, DOI: 10.1016/j.jprot.2018.05.008 (15) Zhang, Y.; Tao, Y.; Zhang, H.; Wang, L.; Sun, G. Q.; Sun, X.; Erinle, K. O.; Feng, C. C.; Song, Q. X.; Li, M. Effect of di-n-butyl phthalate on root physiology and rhizosphere microbial community of cucumber seedlings. J. Hazard. Mater. 2015, 289, 9−17. (16) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72 (1−2), 248−254. (17) Gai, Z.; Zhang, J.; Li, C. Effects of starter nitrogen fertilizer on soybean root activity, leaf photosynthesis and grain yield. PLoS One 2017, 12 (4), No. e0174841. (18) Du, Z.; Zhou, X.; Ling, Y.; Zhang, Z.; Su, Z. agriGO: a GO analysis toolkit for the agricultural community. Nucleic Acids Res. 2010, 38, W64−W70. (19) Xie, C.; Mao, X.; Huang, J.; Ding, Y.; Wu, J.; Dong, S.; Wei, L.; et al. KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Res. 2011, 39, W316− W322. (20) Liu, H.; Sultan, M. A. R. F.; Liu, X. I.; Zhang, J.; Yu, F.; Zhao, H. X. Physiological and comparative proteomic analysis reveals different drought responses in roots and leaves of drought-tolerant wild wheat (Triticum boeoticum). PLoS One 2015, 10 (4), No. e0121852. (21) Wang, S. Q.; Pan, D. Z.; Lv, X. J.; Song, X. M.; Qiu, Z. M.; Huang, C. M.; Huang, R. H.; Chen, W. Proteomic approach reveals that starch degradation contributes to anthocyanin accumulation in tuberous root of purple sweet potato. J. Proteomics 2016, 143, 298− 305. (22) Marschner, P. Rhizosphere biology. In Marschner’s Mineral Nutrition of Higher Plants, 3rd ed.; Elsevier, 2012; pp 369−388. (23) Diaz-Mendoza, M.; Velasco-Arroyo, B.; Santamaria, M. E.; González-Melendi, P.; Martinez, M.; Diaz, I. Plant senescence and proteolysis: two processes with one destiny. Genet. Mol. Biol. 2016, 39 (3), 329−338.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b04956. Venn diagram illustrating the expression patterns (Figure S1), COG analyses (Figure S2), subcellular localization (Figure S3), and GO and KEGG enrichment analyses (Figure S4) of DBP-RPs in the two cultivars and qPCR (Figure S5) and Western blot (Figure S6) verification of some selected proteins (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86 2085223405. Fax: +86 2085226615. ORCID

Ce-Hui Mo: 0000-0001-7904-0002 Zhenli He: 0000-0001-7761-2070 Author Contributions

H.-M.Z. and C.-H.M. designed the research and wrote the manuscript; H.-M.Z., H.-B.H., H.D., Y.-M.L., C.-Q.H., and Y.W.L. performed the experiments and contributed to the data interpretation; L.X., Q.-Y.C., H.L., and Z.H. revised the manuscript for important intellectual content and provided technical and/or material support. Author Contributions §

H.-M.Z. and H.-B.H contributed equally to this work.

Funding

This work was partly funded by the National Natural Science Foundation of China (41703085), the NSFC-Guangdong Joint Fund (U1501233), the Research Team Project of the Natural Science Foundation of Guangdong Province (2016A030312009), the China Postdoctoral Science Foundation (2018T110924), the Natural Science Foundation of Guangdong Province (2017A030313230), the Program of the Guangdong Science and Technology Department (2016B020242005), and the Project of the Guangzhou Science and Technology (201704020074). Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Lü, H.; Mo, C. H.; Zhao, H. M.; Xiang, L.; Katsoyiannis, A.; Li, Y. W.; Cai, Q. Y.; Wong, M. H. Soil contamination and sources of phthalates and its health risk in China: A review. Environ. Res. 2018, 164, 417−429. (2) Niu, L.; Xu, Y.; Xu, C.; Yun, L. X.; Liu, W. P. Status of phthalate esters contamination in agricultural soils across China and associated health risks. Environ. Pollut. 2014, 195, 16−23. (3) Wang, J.; Chen, G. C.; Christie, P.; Zhang, M. Y.; Luo, Y. M.; Teng, Y. Occurrence and risk assessment of phthalate esters (PAEs) in vegetables and soils of suburban plastic film greenhouses. Sci. Total Environ. 2015, 523, 129−137. (4) Sun, J. Q.; Wu, X. Q.; Gan, J. Uptake and metabolism of phthalate esters by edible plants. Environ. Sci. Technol. 2015, 49 (14), 8471−8478. (5) Zhao, H. M.; Du, H.; Xiang, L.; Chen, Y. L.; Lu, L. A.; Li, Y. W.; Li, H.; Cai, Q. Y.; Mo, C. H. Variations in phthalate ester (PAE) accumulation and their formation mechanism in Chinese flowering cabbage (Brassica parachinensis L.) cultivars grown on PAEcontaminated soils. Environ. Pollut. 2015, 206, 95−103. (6) Liu, W.; Zhou, Q.; Li, X. Systematic Selection and Identification of Vegetable Cultivars with Low Heavy Metal Accumulation and for J


Article

Journal of Agricultural and Food Chemistry (24) Sharma, S. S.; Dietz, K. J.; Mimura, T. Vacuolar compartmentalization as indispensable component of heavy metal detoxification in plants. Plant, Cell Environ. 2016, 39 (5), 1112−1126. (25) Sang, Q. Q.; Shan, X.; An, Y. H.; Shu, S.; Sun, J.; Guo, S. R. Proteomic analysis reveals the positive effect of exogenous spermidine in tomato seedlings’ response to high-temperature stress. Front. Plant Sci. 2017, 8, 120. (26) Guo, Z. F.; Tan, J. L.; Zhuo, C. L.; Wang, C. Y.; Xiang, B.; Wang, Z. Y. Abscisic acid, H2O2 and nitric oxide interactions mediated cold-induced S-adenosylmethionine synthetase in Medicago sativa subsp. falcata that confers cold tolerance through up-regulating polyamine oxidation. Plant Biotechnol. J. 2014, 12 (5), 601−612. (27) Ding, W.; Smulan, L. J.; Hou, N. S.; Taubert, S.; Watts, J. L.; Walker, A. K. s-Adenosylmethionine levels govern innate immunity through distinct methylation-dependent pathways. Cell Metab. 2015, 22 (4), 633−645. (28) Stone, S. L. The role of ubiquitin and the 26S proteasome in plant abiotic stress signaling. Front. Plant Sci. 2014, 5, 135. (29) Pommier, Y.; Barcelo, J. M.; Rao, V. A.; Sordet, O.; Jobson, A. G.; Thibaut, L.; Miao, Z. H.; Seiler, J. A.; Zhang, H.; Marchand, C.; Agama, K.; et al. Repair of topoisomerase I-mediated DNA damage. Prog. Nucleic Acid Res. Mol. Biol. 2006, 81, 179−229. (30) Sasikumar, A. N.; Perez, W. B.; Kinzy, T. G. The many roles of the eukaryotic elongation factor 1 complex. WIREs RNA 2012, 3 (4), 543−555. (31) Gupta, K. J.; Igamberdiev, A. U. Reactive nitrogen species in mitochondria and their implications in plant energy status and hypoxic stress tolerance. Front. Plant Sci. 2016, 7, 369. (32) Hu, W. J.; Chen, J.; Liu, T. W.; Wu, Q.; Wang, W. H.; Liu, X.; Shen, Z. J.; Simon, M.; Wu, F. H.; Pei, Z. M.; Zheng, H. L.; Chen, J. Proteome and calcium-related gene expression in Pinus massoniana needles in response to acid rain under different calcium levels. Plant Soil 2014, 380 (1−2), 285−303. (33) Li, H.; Yan, S.; Zhao, L.; Tan, J.; Zhang, Q.; Gao, F.; Wang, P.; Hou, H.; Li, L. Histone acetylation associated up-regulation of the cell wall related genes is involved in salt stress induced maize root swelling. BMC Plant Biol. 2014, 14 (1), 105. (34) Verbančič, J.; Lunn, J. E.; Stitt, M.; Persson, S. Carbon supply and the regulation of cell wall synthesis. Mol. Plant 2018, 11 (1), 75− 94. (35) Zhou, Q.; Guo, J. J.; He, C. T.; Shen, C.; Huang, Y. Y.; Chen, J. X.; Guo, J. H.; Yuan, J. G.; Yang, Z. Y. Comparative transcriptome analysis between low-and high-cadmium-accumulating genotypes of pakchoi (Brassica chinensis L.) in response to cadmium stress. Environ. Sci. Technol. 2016, 50 (12), 6485−6494. (36) Le Gall, H.; Philippe, F.; Domon, J. M.; Gillet, F.; Pelloux, J.; Rayon, C. Cell wall metabolism in response to abiotic stress. Plants 2015, 4 (1), 112−166. (37) Tenhaken, R. Cell wall remodeling under abiotic stress. Front. Plant Sci. 2015, 5, 771. (38) Rabęda, I.; Bilski, H.; Mellerowicz, E. J.; Napieralska, A.; Suski, S.; Wozny, A.; Krzeslowska, M. Colocalization of low-methylesterified pectins and Pb deposits in the apoplast of aspen roots exposed to lead. Environ. Pollut. 2015, 205, 315−326. (39) Wang, X.; Wang, H.; Wang, J.; Sun, R.; Wu, J.; Liu, S.; Bai, Y.; Mun, J. H.; Bancroft, I.; Cheng, F.; Huang, S.; et al. The genome of the mesopolyploid crop species Brassica rapa. Nat. Genet. 2011, 43 (10), 1035. (40) Eticha, D.; Stass, A.; Horst, W. J. Cell-wall pectin and its degree of methylation in the maize root-apex: significance for genotypic differences in aluminium resistance. Plant, Cell Environ. 2005, 28 (11), 1410−1420. (41) Ji, H.; Wang, Y.; Cloix, C.; Li, K.; Jenkins, G. I.; Wang, S.; Shang, Z.; Shi, Y.; Yang, S.; Li, X. The Arabidopsis RCC1 family protein TCF1 regulates freezing tolerance and cold acclimation through modulating lignin biosynthesis. PLoS Genet. 2015, 11 (9), No. e1005471.

(42) Wang, Y.; Chantreau, M.; Sibout, R.; Hawkins, S. Plant cell wall lignification and monolignol metabolism. Front. Plant Sci. 2013, 4, 220. (43) Huang, Y. Y.; Shen, C.; Chen, J. X.; He, C. T.; Zhou, Q.; Tan, X.; Yuan, J. G.; Yang, Z. Y. Comparative transcriptome analysis of two Ipomoea aquatica Forsk. cultivars targeted to explore possible mechanism of genotype-dependent accumulation of cadmium. J. Agric. Food Chem. 2016, 64 (25), 5241−5250. (44) You, J.; Chan, Z. ROS regulation during abiotic stress responses in crop plants. Front. Plant Sci. 2015, 6, 1092. (45) Capaldi, F. R.; Gratão, P. L.; Reis, A. R.; Lima, L. W.; Azevedo, R. A. Sulfur metabolism and stress defense responses in plants. Trop. Plant Biol. 2015, 8 (3−4), 60−73. (46) Liang, T.; Ding, H.; Wang, G.; Kang, J.; Pang, H.; Lv, J. Sulfur decreases cadmium translocation and enhances cadmium tolerance by promoting sulfur assimilation and glutathione metabolism in Brassica chinensis L. Ecotoxicol. Environ. Saf. 2016, 124, 129−137. (47) Noctor, G.; Mhamdi, A.; Chaouch, S.; Han, Y. I.; Neukermans, J.; Marquez-Garcia, B. E. L. E. N.; Queval, G.; Foyer, C. H. Glutathione in plants: an integrated overview. Plant, Cell Environ. 2012, 35 (2), 454−484. (48) Wang, W.; Vinocur, B.; Shoseyov, O.; Altman, A. Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci. 2004, 9 (5), 244−252. (49) Grover, A. Plant chitinases: genetic diversity and physiological roles. Crit. Rev. Plant Sci. 2012, 31 (1), 57−73. (50) de Zelicourt, A.; Colcombet, J.; Hirt, H. The role of MAPK modules and ABA during abiotic stress signaling. Trends Plant Sci. 2016, 21 (8), 677−685. (51) Sang, Q.; Shan, X.; An, Y.; Shu, S.; Sun, J.; Guo, S. Proteomic analysis reveals the positive effect of exogenous spermidine in tomato seedlings’ response to high-temperature stress. Front. Plant Sci. 2017, 8, 120. (52) Hong, J. H.; Seah, S. W.; Xu, J. The root of ABA action in environmental stress response. Plant Cell Rep. 2013, 32 (7), 971−983. (53) Kim, J. S.; Mizoi, J.; Yoshida, T.; Fujita, Y.; Nakajima, J.; Ohori, T.; Todaka, D.; Nakashima, K.; Hirayama, T.; Shinozaki, K.; Yamaguchi-Shinozaki, K. An ABRE promoter sequence is involved in osmotic stress-responsive expression of the DREB2A gene, which encodes a transcription factor regulating drought-inducible genes in Arabidopsis. Plant Cell Physiol. 2011, 52 (12), 2136−2146. (54) Liu, F.; Lu, C. M. An overview of non-specific lipid transfer protein in plant. Yichuan 2013, 35 (3), 307−314. (55) Ravet, K.; Pilon, M. Copper and iron homeostasis in plants: the challenges of oxidative stress. Antioxid. Redox Signaling 2013, 19 (9), 919−932. (56) Dietz, K. J.; Tavakoli, N.; Kluge, C.; Mimura, T.; Sharma, S. S.; Harris, G. C.; Chardonnens, A. N.; Golldack, D. Significance of the Vtype ATPase for the adaptation to stressful growth conditions and its regulation on the molecular and biochemical level. J. Exp. Bot. 2001, 52 (363), 1969−1980.

K


Differences in Root Physiological and Proteomic Responses to Dibutyl

Recommend Documents