Global Picture of Protein Regulation in Response to Dibutyl Phthalate

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Omics Technologies Applied to Agriculture and Food

Global picture of protein regulation in response to dibutyl phthalate (DBP) stress of two Brassica parachinensis cultivars differing in DBP accumulation Hai-Ming Zhao, He-Biao Huang, Huan Du, Lei Xiang, Ce-Hui Mo, Yan-Wen Li, Quan-Ying Cai, Hui Li, Jie-Sheng Liu, Dong-Mei Zhou, and Ming-Hung Wong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01157 • Publication Date (Web): 23 Apr 2018 Downloaded from http://pubs.acs.org on April 24, 2018

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Journal of Agricultural and Food Chemistry

Global picture of protein regulation in response to dibutyl phthalate (DBP) stress of two Brassica parachinensis cultivars differing in DBP accumulation

Hai-Ming Zhao†, He-Biao Huang†, Huan Du†, Lei Xiang†, Ce-Hui Mo†,*, Yan-Wen Li†, Quan-Ying Cai†, Hui Li†, Jie-Sheng Liu†, Dong-Mei Zhou†,‡, Ming-Hung Wong†,⊥

†

Guangdong Provincial Research Center for Environment Pollution Control and Remediation Materials, School

of Environment, Jinan University, Guangzhou 510632, China ‡

Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of

Sciences, Nanjing 210008, China ⊥

Consortium on Environment, Health, Education and Research (CHEER), The Education University of Hong

Kong, Hong Kong, China

*Corresponding Author Ce-Hui Mo. E-mail: [email protected]. Tel: +86 2085220564. Fax: +86 2085226615.

ACS Paragon Plus 1 Environment


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ABSTRACT: iTRAQ analysis was used to map the proteomes of two Brassica parachinensis

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cultivars that differed in DBP accumulation. A total of 5,699 proteins were identified to obtain

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152 differentially regulated proteins, of which 64 and 48 were specific to a high and a low

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DBP-accumulation cultivar, respectively. Genotype-specific biological processes were involved in

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coping with DBP stress, accounting for the variation in DBP tolerance and accumulation.

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Formation of high DBP accumulation in B. parachinensis might attribute to the more effective

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regulation of protein expression in physiology and metabolism, including (a) enhanced cell wall

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biosynthesis and modification, (b) better maintenance of photosynthesis and energy balance, (c)

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greatly improved total capacity for antioxidation and detoxification, and (d) enhanced cellular

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transport and signal transduction. Our novel findings contribute to a global picture of

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DBP-induced alterations of protein profiles in crops and provide valuable information for the

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development of molecular-assisted breeds of low-accumulation cultivars.

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KEYWORDS: Brassica parachinensis, proteomic, dibutyl phthalate (DBP) stress, accumulation

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variation, low-accumulation cultivars

16 17 18 19 20 21 22 23


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1. INTRODUCTION

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Dibutyl phthalate (DBP) is one of the most widely used phthalate esters (PAEs), resulting in large

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releases into the environment on a global scale.1 It has been listed as a priority pollutant by the

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U.S. Environmental Protection Agency and the Chinese Environment Monitoring Center because

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it is an endocrine disrupting compound with potentially teratogenic, mutagenic, and carcinogenic

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properties even at very low concentrations.2 The extensive use of DBP-containing products has

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lead to ubiquitous contamination of DBP in agricultural soils, and its concentrations are usually

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higher than other individual PAE compounds.3 It was reported that the residual of DBP ranged

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from 3.18 to 29.37 mg/kg in some typical agricultural soils of China, and even up to 57.7 mg/kg

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in cotton field soils.4-6 Notably, the use of large amounts of agricultural plastic film and fertilizers

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containing DBP in intensive vegetable cultivation might increase the human health risks from

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DBP through the food chain.3,7 It has been estimated that the contributing of DBP exposure to

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human through the pathway of vegetable consumption is 42.4%.8 Therefore, employing efficient

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strategies and technologies to minimize DBP accumulation in vegetables is important to reduce

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the pollution risk from DBP in the food chain.

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The application of low-accumulation crop cultivars that contain a low level of pollutants in the

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edible parts is considered a practical and cost-effective approach to minimize the entry of

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pollutants into the human food chain. This approach has received widespread attention.9,10 In

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recent years, many researchers have reported low-accumulation cultivars of heavy metals (e.g.,

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cadmium [Cd], arsenic, copper) including rice,11 wheat,9 pakchoi,10 water spinach,12 Chinese

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cabbage,13 and others. Some of these low-accumulation cultivars have been successfully

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commercialized. For example, peanut (Florunner) and soybean (Soy791) low-Cd accumulation

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cultivars have been recommended for use in Australia.14 The low-Cd accumulation cultivars of



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durum wheat (Strongfield) that was released in 2004 is now sown on more than 25% of the area

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for durum wheat in Canada.9 Furthermore, the physiological, biochemical, and molecular

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mechanisms responsible for low-accumulation cultivars of heavy metals have been

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extensively studied10,12 and some key genes that limit heavy metal (e.g., Cd, arsenic, copper)

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accumulation in rice have been identified.11,15 By contrast, little work has been reported on

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low-accumulation cultivars of organic pollutants, with the exception of our studies on low-PAEs

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accumulation cultivars in Brassica parachinensis and rice.16–18 As typical non-ionic organic

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chemicals, PAEs are quite different from heavy metals in terms of their uptake, accumulation, and

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tolerance.19 Thus, screening and further studying low-accumulation cultivars of organic pollutants,

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such as PAEs, is necessary and urgent to perfect low-accumulation cultivars strategies and ensure

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safe agricultural products that consider the ubiquitous presence of organic contaminants in

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agricultural soils.

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B. parachinensis is one of the most important leafy vegetables in southern China, and

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is exported to many countries and regions around the world. Our previous investigation suggested

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that B. parachinensis accumulated the highest level of ΣPAEs (especially DBP) among the 11

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investigated vegetable species collected from the Pearl River Delta area in southern China, and its

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bioconcentration factors for PAEs were also generally higher than other vegetables.3 Moreover,

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the bioconcentration of PAEs in B. parachinensis depended on genotypes, showing a stable

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biological property at cultivar level.17 Hence, we screened two cultivars of B. parachinensis that

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accumulated high DBP (Huaguan) and low DBP (Lvbao) levels, and the physiological mechanism

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regulating the variation in DBP accumulation in the two cultivars has been preliminarily

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studied.17,18 However, the molecular basis of such a physiological trait variation between the two

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cultivars remains unknown. The intricacies of such complex regulatory mechanisms require


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global analysis of genes, proteins, and metabolites to identify the interconnected networks

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regulating DBP uptake, accumulation, and tolerance under DBP stress.

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Because gene expression changes ultimately result in changes in the expression levels or

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activities of various proteins, a comprehensive understanding of DBP-responsive proteomic

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changes is also warranted to fully appreciate the scope of such global analyses. As the functional

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translated portion of the genome plays a key role in the plant stress response, proteomic studies

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provide a finer picture of the protein networks and metabolic pathways primarily involved in the

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internal regulation of physiological processes at the molecular level.20,21 Compared with the

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genomic study, the proteomic data permit different information that is complementary to clarify

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the biochemical responses as well as to deep the knowledge regarding regulatory mechanisms in

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biological processes. Recently, an iTRAQ-based quantitative proteomic approach has been widely

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used for the comparative analyses of proteome changes because it allows for the simultaneous

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identification and quantification of peptides by measuring the peak intensities of reporter ions

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with tandem mass spectrometry (MS/MS).22 This approach is advantageous because it can reveal

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the underlying cellular processes of stress-response relationships between expression levels of the

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differential proteins and plant tolerances to pollutants using iTRAQ-based proteomics.20-22

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However, until now, no investigations on plant responses to organic pollutant stress (especially

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PAEs) have been conducted using this technique. Thus, in this study, iTRAQ-based analysis of

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proteome variation in the two B. parachinensis cultivars in response to DBP stress may provide

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an important information regarding DBP-stress related proteins and the metabolic pathways

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regulating the physiological processes of DBP uptake and accumulation.

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In this study, iTRAQ technology was first used to determine the molecular mechanisms that

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differed in the proteomic responses to DBP stress of two B. parachinensis cultivars (high- and



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low-DBP accumulators), and to identify differentially regulated proteins (DRPs) involved in the

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variation of DBP accumulation between the two cultivars exposed to DBP. Two major issues are

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expected to clarify by comparative proteomic analysis: (1) what are the differences in the

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proteomic responses to DBP stress associated with different cultivars; and (2) what is the genetic

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basis for the different capabilities of DBP accumulation between the two cultivars. At the

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proteomic level, this study can provide new insights into the molecular mechanisms responsible

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for high/low-DBP accumulation in various cultivars of B. parachinensis. This will be of great

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significance to the evolution of standard methodology for molecular breeding for

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low-accumulation crop cultivars via genetic improvement.

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2. MATERIALS AND METHODS

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2.1. Plant cultivation and DBP treatments. A hydroponic experiment was carried out in

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a greenhouse at 25-32°C (day) and 22-26°C (night) under natural light conditions. The

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hydroponic solution prepared in 1.0 L of water contained: NH4NO3 (80 mg), KNO3 (202 mg),

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Ca(NO3)·4H2O (472 mg), KH2PO4 (100 mg), MgSO4·7H2O (246 mg), K2SO4 (174 mg),

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FeSO4·7H2O (27.8 mg), ZnSO4·7H2O (0.22 mg), EDTA-2Na (37.2 mg), CuSO4·5H2O(0.08 mg),

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MnSO4·4H2O (2.13 mg), H3BO3 (2.86 mg), and (NH4)6MO7O24·4H2O (0.02 mg). Two cultivars

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of B. parachinensis with high (Huaguan) and low (Lvbao) DBP accumulation were chosen as the

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experimental plants based on our previous study.17 Based on the results of our pre-experiment

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(DBP-tolerance test, please see Supporting Information), the DBP concentration at 25 mg/L was

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selected as a representative concentration to investigate protein profiles of the two cultivars

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differing in DBP accumulation, which was a mild stress condition that could synchronously

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ensure a larger difference in DBP tolerance between the two cultivars and a greater proportion of

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tolerant individuals of the two cultivars (Figure S1 and Table S1). After seed germination in ACS Paragon Plus 6 Environment

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vermiculite for 2 weeks, uniform seedlings with 4-5 leaves were selected and transplanted into

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aerated hydroponic solutions. The seedlings were grown in hydroponic solution without (control)

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or with 25 mg/L DBP (treatment). A stock methanol solution of DBP was added to the hydroponic

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solution according to the method described by Kang et al.23 The methanol concentration in the

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hydroponic solution was less than 0.1% (v/v) and was also added in control. Hydroponic solutions

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with and without DBP were renewed every two days, respectively. After 15 days of growth, plants

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from the two tested cultivars were harvested and washed three times with deionized water. The

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fresh plants were separated into shoots and roots and weighed. Fresh young leaves were

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immediately frozen in liquid nitrogen and stored at –80°C for protein extraction. The young

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leaves of five plants were collected as one independent biological replicate, and four biological

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replicates were combined into one sample to generate a sufficient sample quantity for one

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technical replicate. A total of two technical replicates were analyzed using an 8-plex iTRAQ

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labeling experiment.

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2.2. Analysis of DBP in shoots. Shoot samples of the two cultivars were freeze-dried at

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-55 °C and ground to pass through a stainless-steel sieve (0.4 mm). We performed

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ultrasonic-assisted sample extraction, cleanup using a silica gel column, and DBP analysis using

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gas chromatography coupled with mass spectrometry (GC/MS, Shimadzu QP2010 Plus, Japan) as

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described in our previous reports.17,18 The detection limit of DBP was 1.2 µg/kg. The recoveries of

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DBP in the shoot samples ranged from 87.4 to 107.2%. Representative GC/MS chromatograms of

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the tested DBP were provided in Supporting Information (Figure S2).

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2.3. Leaf protein extraction and trypsin digestion. The leaf samples were ground with

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liquid nitrogen and the cell powder was transferred to a 5 mL centrifuge tube and sonicated three

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times on ice using a high intensity ultrasonic processor in lysis buffer (8 M urea, 1% Triton-100,



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65 mM DTT, 2 mM EDA and 1% Protease Inhibitor Cocktail, pH 8.0). The remaining debris was

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removed by centrifugation at 20,000 × g at 4°C for 10 min. Finally, the protein was precipitated

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with cold 15% trichloroacetic acid for 2 h at –20°C. After centrifugation at 20,000 × g at 4°C for

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10 min, the supernatant was discarded. The remaining precipitate was washed with cold acetone

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three times. The protein was re-dissolved in the buffer solution (8 M urea, 100 mM TEAB, pH 8.0)

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and the protein concentration was determined using a 2-D Quant kit (GE Healthcare, USA)

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according to the manufacturer’s instructions. Approximately 100 µg of protein for each sample

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was digested with trypsin (Promega, USA) at 37°C in a 1:50 trypsin-to-protein mass ratio for the

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first digestion overnight and a 1:100 trypsin-to-protein mass ratio for a second digestion lasting

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4h.

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2.4. iTRAQ labeling and high-performance liquid chromatography (HPLC)

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fractionation. After the trypsin digestion, the peptides were desalted using a Strata X C18 SPE

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column (Phenomenex, USA) and vacuum-dried. The peptides were reconstituted in 0.5 M

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triethylammonium bicarbonate and processed according to the manufacturer’s protocol for the

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8-plex iTRAQ kit (AB SCIEX, USA). For each cultivar (i.e., Huaguan or Lvbao), four samples

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(two technical replicates with eight biological replicates for the control and DBP treatments,

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respectively) were iTRAQ labeled: 113-, 114-, 117-, and 118-iTRAQ tags for the control and

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DBP-treated replicate 1 and replicate 2 in Huaguan; and 115-, 116-, 119- and 121-iTRAQ tags for

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the control and DBP-treated replicate 1 and replicate 2 in Lvbao. Briefly, one unit of iTRAQ

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reagent (defined as the amount of reagent required to label 100 µg of protein) was thawed and

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reconstituted in 24 µl acetonitrile. The iTRAQ-labeled peptide mixtures were then incubated for 2

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h at room temperature and pooled, desalted and dried by vacuum centrifugation.

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The pooled peptides were fractionated using high pH reverse-phase HPLC (Agilent 1260 series,


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USA) and an Agilent 300 Extend C18 column (5 µm particles, 4.6 mm ID, 250 mm length).

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Briefly, the peptides were first separated with a gradient of 2% to 60% acetonitrile in 10 mM

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ammonium bicarbonate pH 10 over 80 min into 80 fractions, Then, the peptides were combined

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into 18 fractions and dried by vacuum centrifuging.

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2.5. Quantitative proteomic analysis by LC-MS/MS. The peptides were dissolved in

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solvent A (0.1% formic acid) and directly loaded onto a reversed-phase pre-column (Acclaim

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PepMap 100, Thermo Fisher Scientific, USA). Peptide separation was performed using a

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reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Fisher Scientific). The

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gradient included solvent B (0.1% formic acid in 98% acetonitrile) which increased from 7 to

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20% in 24 min, 20 to 35% in 8 min, and climbed to 80% in 4 min; it then held at 80% for the last

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4 min, all at a constant flow rate of 300 nl/min on an EASY-nLC 1000 UPLC system (Thermo

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Fisher Scientific). The resulting peptides were analyzed using a Q ExactiveTM Hybrid

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Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific).

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The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q

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ExactiveTM (Thermo) coupled online to the UPLC. Intact peptides were detected in the Orbitrap at a

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resolution of 70,000. Peptides were selected for MS/MS using NCE setting as 32; ion fragments

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were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated

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between one MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions above

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a threshold ion count of 1E4 in the MS survey scan with 30.0s dynamic exclusion. The electrospray

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voltage applied was 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the

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ion trap; 5E4 ions were accumulated for generation of MS/MS spectra. For MS scans, the m/z scan

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range was 350 to 1800. Fixed first mass was set as 100 m/z. The accuracy of MS data was validated

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by checking the distributions of mass error and peptide length. The distribution of mass error was



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near zero and most of them were less than 3 ppm, which suggested the mass accuracy of the MS

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data met the requirement (Figure S3a). The lengths of most peptides distributed between 8 and 16,

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which agreed with the property of tryptic peptides, suggesting that sample preparation reached the

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standard (Figure S3b). Representative LC-MS/MS chromatogram of the proteomes quantification

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was provided in Supporting Information (Figure S4).

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2.6. Bioinformatics analysis. The raw MS/MS data files acquired from Orbitrap were

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processed with a thorough database search against the Chinese cabbage (B. pekinensis) Uniprot

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database using MaxQuant software version 1.5.3.8. The digestion enzyme was set to trypsin/P

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with up to two missed cleavages. “Ubiquitylation” (GlyGly [K]), “oxidation” (M), and

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“N-terminal acetylation” were searched as variable modifications and “carbamidomethyl” (C), the

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iTRAQ 8 plex (N-term), as used as a fixed modification. Unused ProtScore >1.3 as threshold with

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at least more than one peptide above the 95% confidence level was considered as benchmark for

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protein identification, which ensured the accuracy of change trend in protein expression. A ratio of

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fold change > 1.5 was set to determine DRPs with a t-test p-value < 0.05. AgriGO

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(http://bioinfo.cau.edu.cn/agri GO/index.php) was used for the annotation and functional

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classification of identified proteins according to the Gene Ontology (GO) and the UniProt

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databases. We also conducted a Cluster of Orthologous Groups of proteins (COG) analysis

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(http://www.geneontology.org). KOBAS 2.0 (http://kobas.cbi.pku.edu.cn/) was used for the

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protein pathway analysis according to the Kyoto Encyclopedia of Genes and Genomes Pathway

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Database. Hierarchical clustering of protein abundance data was further processed using Gene

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Cluster software, version 3.0.

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2.7. Quantitative polymerase chain reaction (qPCR) analysis. To investigate whether

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gene expression was correlated between the transcript and protein level and confirm the


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DBP-response proteins in the iTRAQ data set, the expression changes of 12 selected genes were

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analyzed by qPCR experiment. The sequences for the gene-specific primers are provided in

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Supplementary Table S2. The actin gene was used as an internal control. Total RNA was extracted

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from DBP-treated and control plant leaves with three independent biological replicates by

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RNAiso Plus (Takara, China). First strand cDNA was generated by purified total RNA using a

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PrimeScript RT regent kit. SYBR Green PCR cycling was performed on a CFX9600 Real-Time

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PCR system (Bio-Rad, USA) using 20 µL samples according to the manufacturer's instructions.

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The analysis of each sample was performed in triplicate, and the relative quantification of the

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expression of each gene was calculated and normalized to actin using the 2− ∆∆CT method. 24

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3. RESULTS AND DISCUSSION

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3.1. Variation in shoot growth and DBP concentration between the two cultivars.

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Both shoot biomass and DBP concentration in Lvbao were significantly lower (p < 0.05) than

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those in Huaguan when exposed to DBP (Figure 1a and Table S3). Additionally, the leaves of

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Lvbao exhibited abnormally yellow spotting (Figure 1b), indicating that DBP-stress-induced

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inhibition of shoot growth was more severe in Lvbao than in Huaguan.

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3.2. Overview of quantitative proteomics analysis. A total of 178,164 spectra were

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generated from the iTRAQ experiment using DBP-treated and untreated shoots of two B.

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parachinensis cultivars. By analyzing these spectra using MaxQuant software, 30,503 known

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spectra, 13,568 peptides, 7,963 unique peptides, and 5,699 proteins were identified (Figure S5a).

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Protein number decreased with an increased number of peptides that were matched to proteins,

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but over 62% of the proteins included at least two peptides (Figure S5b). The protein mass

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distribution suggested that a good coverage was obtained for a wide range of molecular weights

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for proteins larger than 10 kDa (Figure S5c). Additionally, most of the proteins were identified ACS Paragon Plus 11 Environment


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with good sequence coverage, and over 50% of the proteins constituted more than 10% of the

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sequence coverage, indicating high confidence (Figure S5d). Gene Ontology (GO) function

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classification analysis of all the quantified proteins used three different criteria of protein

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functional annotation, including biological process, cellular component, and molecular function

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(Figure S6). This suggested that these proteins were involved in almost every aspect of plant

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metabolism, and may play important roles in B. parachinensis’ response to DBP stress.

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3.3. Differentially regulated proteins (DRPs) regulated by DBP stress. Using cutoffs

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of both a fold change > 1.5 and a p-value < 0.05 compared to the untreated control plants, 152

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proteins were observed with significant changes in the two cultivars when exposed to DBP stress

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(Table S4): 104 proteins were identified in the high-DBP cultivar (Huaguan), 88 proteins were

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identified in the low-DBP cultivar (Lvbao), and 40 proteins were shared between both cultivars

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(Figure 2a). The results indicated that different strategies might be adopted in response to DBP

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stress between the high- and low-DBP cultivars. Furthermore, it was clear that there were more

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DRPs identified in Huaguan than in Lvbao. This demonstrated that the metabolic flexibility was

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stronger in Huaguan than in Lvbao in response to DBP stress, considering the higher DBP

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accumulation and tolerance of the former. To further elucidate the global function of proteins

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responsive to DBP stress, the DRPs were categorized into different functional groups based on the

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COG database. Except for the general function prediction, which was only 9.7%, the percentages

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of replication, recombination, and repair (9.2%), post-translational modification, protein turnover,

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and chaperones (7.9%), transcription (7.2%), and signal transduction mechanisms (7.2%) were

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dominant, indicating that posttranscriptional regulation played an important role in the

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responsiveness of B. parachinensis to DBP stress (Figure 2b).

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Cluster analysis of the DRPs based on the iTRAQ data demonstrated high reproducibility in


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relative protein abundance in DBP-treated plants vs. the control between two replicates (Figure 3).

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Hierarchical clustering of DRPs occurred only in Huaguan in response to DBP stress with more

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upregulated (Cluster I) than downregulated proteins (Cluster II) (Figure 3a). However, a similar

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situation was insignificant in Lvbao (Figure 3b). This showed that the regulatory mechanism for

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protein expression between the two cultivars when exposed to DBP stress differed significantly.

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Cluster analysis of the 40 co-regulated proteins in the two cultivars in response to DBP revealed

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that the DRPs could be divided into three groups (Figure 3c). Overall, these results indicated that

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most DRPs were upregulated under DBP stress in the leaves of the two cultivars.

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3.4. Validation of differentially regulated proteins (DRPs) by qPCR. To validate the

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reliability of the data from the iTRAQ analysis, 12 proteins selected from the 40 co-regulated

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DRPs in the two cultivars were subjected to qPCR analysis. Most of the mRNA expression levels

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of the selected proteins showed similar trends as in the iTRAQ results, except for two proteins:

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thaumatin-like protein and LEA family protein (Figure S7). After removing the quantitative

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values of the two proteins, the qPCR results showed a strong correlation with the iTRAQ data (R2

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= 0.85, Figure 4), suggesting that the regulation of these proteins likely resulted from

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transcriptional induction of the corresponding genes in the leaves of the two cultivars under DBP

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stress. However, the discrepancy in mRNA expression levels in the two proteins was also found in

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other proteomic studies, which might be attributed to post-transcriptional and translational

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regulatory processes or feedback loops between the processes of mRNA translation and protein

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degradation.25

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3.5. Enhanced cell wall biosynthesis and modification promoted DBP tolerance

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and accumulation. Plant cell walls protect protoplasts against contaminant toxicity by binding

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organic contaminants via cell wall components such as cellulose, pectin, hemicellulose, and lignin,



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thus restricting transport of the contaminants across cell membranes (Figure 5).23 Our previous

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study revealed that PAEs mainly accumulated in the cell walls of B. parachinensis with

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significantly higher DBP concentrations in Huaguan than in Lvbao.18 In this study, over 20% of

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the total DRPs were involved in processes linked to cell wall biosynthesis and modification, and

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most of them were upregulated in Huaguan (Table S4), indicating that cell wall processes are one

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of the key factors determining the genotype dependent tolerance and accumulation of DBP in

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shoots of the two cultivars at the proteomic level.

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Cellulose is a main load-bearing wall component synthesized by cellulose synthases.26 The

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cellulose synthase proteins that catalyze the biosynthesis of cellulose from uridine

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5′-diphosphoglucose (UDP-glucose) were significantly upregulated in Huaguan (Figure 5).

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Increased cellulose synthesis could maintain cell wall integrity and turgor pressure in Huaguan,

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thus allowing continuous cell growth under environmental stress.10,27 On the other hand,

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endoglucanases and β-glucosidases could play important roles in development, remodeling, and

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degradation of plant cell walls,28 and were upregulated only in Lvbao (Figure 5), suggesting a

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different mechanism of cell wall response to DBP stress between the two cultivars, which

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deserves further study.

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Hemicelluloses can strengthen cell walls by interaction with cellulose and/or lignin and are

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mainly comprised of xyloglucans and xylans in most dicots.28 These enzymes are related to

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xyloglucan and xylan synthesis and are involved in the metabolic pathways of glucose and

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fructose. They were upregulated more obviously in Huaguan than in Lvbao (Figure 5), indicating

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a more massive deposition of hemicelluloses inside the primary wall of Huaguan. This, together

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with increased cellulose, suggests an internal mechanism and the basis for the increased thickness

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of the cell wall in Huaguan as observed by transmission electron microscopy.18 Furthermore, cell


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wall modification is an important aspect of plant acclimation to environmental stresses.28

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Structural changes to the existing cell wall mediated by various cell wall modifying proteins help

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plants adapt to environmental stresses by regulating cell growth. The hemicellulose

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(xyloglucan)-cellulose network forms the major tension-bearing structure in plant cell walls

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during

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endotransglucosylases/hydrolases (XTHs). XTHs can not only strengthen cell walls via the

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integration of newly secreted xyloglucans into the cell wall, but can also remodel cell walls via

307

cutting and rejoining of xyloglucan chains.28 In this study, more XTHs were significantly

308

upregulated in Huaguan than in Lvbao, indicating that cell wall modification was more active in

309

the high-DBP cultivar under DBP stress. Similar results concerning upregulated cell wall

310

biosynthesis and modification genes encoding cellulose synthases and XTHs were observed in

311

Sedum alfredii Hance, a heavy metal hyperaccumulator.29

cellular

expansion,

which

is

the

target

for

proteins

called

xyloglucan

312

Pectin is a class of galacturonic acid containing polysaccharides that forms a hydrated gel-like

313

matrix in which cellulose and hemicelluloses are embedded.30 Pectins are deposited in highly

314

esterified forms in the cell wall as substrates for groups of cell wall-modifying enzymes, such as

315

pectinesterase, polygalacturonase, pectate lyase, and pectin acetylesterase.28 It has been reported

316

that the degree of methylesterification of pectins has a great effect on heavy metal tolerance and

317

accumulation.28 Low-methylesterified pectins contain some free carboxyl groups and provide

318

negatively-charged binding sites in the cell wall, which is conducive to adsorbing and

319

accumulating various pollutants. In this study, two pectin-modifying enzymes, pectinesterase and

320

polygalacturonase, which can catalyze the demethylesterification of pectins, were only

321

significantly upregulated in Huaguan (Figure 5), suggesting an enhanced adsorption affinity of

322

the cell wall for DBP. However, pectate lyases were clearly upregulated in Lvbao (Figure 5), and



323

therefore may play important roles in the activation of defense systems through the release of

324

oligogalacturonides or premature flowering as observed in this study by stimulating pollen tube

325

growth.31

326

Lignin derived from the phenylpropanoid biosynthesis pathway is the second most abundant

327

plant biopolymer after cellulose, and it responds to many developmental and environmental

328

cues.32 For developmentally programmed deposition of lignin, most of which has been observed

329

in association with secondary wall thickenings and programmed cell death.33 Besides, its

330

biosynthesis can also be induced upon various biotic and abiotic stress conditions.32 The enzymes

331

participating in lignin synthesis were significantly upregulated in both cultivars (Figure 5),

332

indicating that lignification as a protective response could occur prematurely to avoid cell wall

333

damage under DBP stress.28 This was supported by the results of the transcriptome analysis in B.

334

chinensis where Cd stress induced lignification in roots by regulation of the phenylalanie

335

metabolic pathway.10 Thus, the higher percentages of upregulated proteins related to lignin

336

synthesis (Table S4) in Huaguan might be responsible for its higher DBP tolerance compared to

337

Lvbao. Overall, DBP-induced proteomic changes in cell wall biosynthesis and modification could

338

be important molecular processes responsible for the genotype variation in DBP tolerance and

339

accumulation in B. parachinensis, which needs to be further investigated urgently.

340

3.6. Differential regulation in DRPs linked to basic metabolic processes between

341

the two cultivars. Our previous study on bio-physiology showed that DBP could clearly inhibit

342

photosynthesis in both cultivars, especially for Lvbao.18 In this study, expression of most of the

343

predicated proteins involved in photosynthesis was significantly downregulated in the two

344

cultivars under DBP stress, especially in Lvbao (Table S4). The light-harvesting chlorophyll

345

a/b-binding (LHC) proteins function in photosystem (PS) I and II as coordinators of antenna


Page 16 of 38

Page 17 of 38


346

pigments, which play an important role in light harvesting. Decreased expression of LHC proteins

347

resulted in impaired stacking of grana in the chloroplasts of Arabidopsis.34 Accordingly, the

348

downregulation of LHC proteins was responsible for abnormal chloroplast development and

349

decreased chlorophyll content in the two cultivars, as observed previously.18 Furthermore, of the

350

12 downregulated DRPs related to the chlorophyll biosynthesis pathway, more of them occurred

351

in Lvbao than in Huaguan, and three DRPs involved in chlorophyll transformation were

352

upregulated in Lvbao only (Table S4 and Figure 6), which accounted for the greater decrease in

353

chlorophyll content and etiolation in Lvbao, as observed previously.18 The results implied that

354

greater downregulation or disruption of DRPs involved in photosynthesis in the low-DBP cultivar

355

might result in a greater decrease in tolerance to DBP compared with the high-DBP cultivar.

356

Many studies have shown that DBP has adverse effects on plant development, such as massive of

plant

cellular

structures.18,35

357

destruction

In

this

study,

358

cytoskeleton-related proteins, actin and profilin, in the two cultivars (Table S4) might be partially

359

responsible for the damage or disorder imparted to cell development. However, two other proteins,

360

myosin and tubulin, which are involved in modulating the properties and/or functions of the actin

361

filament and constructing microtubule skeletal structures, the stress response, etc.,25,36 were

362

upregulated in Huaguan with no change in Lvbao (Table S4), suggesting that they might play

363

distinct roles in the regulation of cell growth and development in Huaguan by enhancing adaption

364

to DBP stress. The upregulation of other proteins concerning plant development and response to

365

abiotic stress could also enhance plant resistance to toxicants.21 In this study, the DNA

366

repair-related proteins were upregulated to a greater extent in Huaguan than in Lvbao and the

367

TRAF-like protein was upregulated in Huaguan but downregulated in Lvbao (Table S4), which

368

also accounted for, to some extent, the higher tolerance of the former to DBP toxicity.


downregulation

of

the


369

A general symptom of photosynthetic plants under stress is energy deficit caused by a

370

reduction in photosynthesis and/or respiration, ultimately resulting in growth arrest and cell

371

death.37 Plant ATP synthases located in different subcellular organelles have different functions

372

including proton transport, regulation of intracellular pH redox homeostasis, stomatal opening,

373

plant growth, and the stress response.25 In this study, a decrease in ATP synthase subunit delta

374

proteins in both of the cultivars suggested that ATP synthesis might be suppressed by DBP, thus

375

restricting growth and development. The ATP synthase subunit beta protein was also

376

downregulated in Lvbao with little change in Huaguan (Table S4), which suggested a more

377

specific response to the inhibition of ATP synthesis in low-DBP cultivars under DBP stress.

378

Moreover, adenylate kinase, which is related to cellular energy homeostasis and adenine

379

nucleotide metabolism,38 was clearly upregulated in Huaguan. All of these results implied that

380

Huaguan could operate the related biological processes well enough to resist DBP toxicity by

381

maintaining ATPases at a higher level compared to Lvbao.

382

3.7. More active DRPs regulate stress tolerance and defense in the high-DBP

383

cultivar. Plants have tolerance and defense mechanisms to respond to abiotic stress and aid in

384

survival.39 A set of 52 proteins that accounted for 34% of the DRPs belonged to this category

385

(Table S4). The generation of reactive oxygen species (ROS) is one of the first universal reactions

386

to DBP toxicity in plants, caused by many oxidation reactions that are activated at the onset of

387

environmental stress.36 During a defense response to DBP toxicity, many detoxification related

388

proteins, including cytochrome P450s, peroxidases, and lipoxygenases with the putative function

389

of generating ROS, were detected in our iTRAQ data (Table S4 and Figure 6). Oxidative

390

metabolism mediated by P450s and peroxidase is particularly important for the detoxification of

391

xenobiotics in a variety of crop species.40 Lipoxygenase is involved in the activation of membrane


Page 18 of 38

Page 19 of 38


392

lipid peroxidation by the oxidation of polyunsaturated fatty acids to ROS.25 In this study, the

393

abundance of these proteins in both cultivars was clearly regulated under DBP stress, indicating

394

that the detoxification metabolism of DBP was likely activated. More pronounced upregulation of

395

the detoxification related proteins in Huaguan might be responsible for its higher DBP tolerance

396

compared with Lvbao. However, the exact mechanisms of detoxification of DBP remain unknown

397

and require further study.

398

Antioxidant enzymes are known to play important roles in scavenging or reducing excessive

399

ROS to maintain cell redox homeostasis.41 Here, we identified a group of antioxidant enzymes,

400

including catalase, superoxide dismutase, ascorbate peroxidase, and glutathione S-transferase

401

(GST) (Table S4). Decreased levels of catalase and superoxide dismutase in the two cultivars

402

indicated a loss of protective capacity against cellular ROS toxicity, owing to a transient shortage

403

of catalase and superoxide dismutase during their consumption over the short term.18 Furthermore,

404

more pronounced decreases of catalase, superoxide dismutase, and ascorbate peroxidase in Lvbao

405

suggested the breakdown of the antioxidant system, thus partly resulting in its low tolerance to

406

DBP. By contrast, higher levels of ascorbate peroxidases and GSTs in Huaguan assisted in

407

minimizing oxidative damage, as they enabled a greater capacity to resist DBP stress.

408

Furthermore, it has been reported that GSTs can detoxify a variety of xenobiotics by catalyzing

409

conjugation of the reduced glutathione (GSH) with these xenobiotics to yield less toxic

410

metabolites.42 GSH is the most abundant intracellular non-protein thiol, and is involved in many

411

cellular functions, including DNA repair, redox regulation, and multiple cell signaling pathways.43

412

Both cysteine and glutamate are important precursors of GSH biosynthesis. Similarly, sulfur

413

metabolism in plants is a core pathway for cysteine synthesis. During this process, the related

414

enzymes play key roles in the development of tolerance to pollution stresses.10,21 In this study,



415

sulfur metabolism-related enzymes such as ATP sulfurylase, sulfite reductase, cysteine ligase, and

416

glutathione synthase were more demonstrably upregulated in Huaguan than in Lvbao (Table S4

417

and Figure 6), indicating stronger sulfur metabolism in the former. Similarly, a pathway of

418

nitrogen metabolism involved in glutamate synthesis was also activated, in which two key

419

enzymes, glutathione synthase and glutamate synthase (GOGAT), were upregulated in the two

420

cultivars, especially in Huaguan. Moreover, overexpression of these two enzymes could enhance

421

the proline content in plant leaves, assisting in ameliorating environmental stress. On the other

422

hand, three upregulated glutathione reductases and two downregulated glutathione peroxidases

423

might keep a favorable GSH/glutathione disulfide (GSSG) ratio for the detoxification of DBP in

424

Huaguan.44 But the opposite trend appeared in Lvbao, resulting in low tolerance to DBP.

425

Moreover, in the pentose phosphate pathway, glucose-6-phosphate dehydrogenase catalyzed the

426

key NADPH-production step that is known to play a role in the protection against oxidative

427

stress.9 In this study, glucose-6-phosphate dehydrogenase was upregulated in Huaguan with little

428

change in Lvbao (Figure 6), suggesting greater antioxidative properties in the former.

429

Heat shock proteins (HSPs) are the most common protective proteins that respond to stress

430

stimuli in most cells and can mediate the correct folding of proteins to prevent further damage and

431

repair intracellular injury.25 HSP70 can prevent proteins from degradation and aggregation, and it

432

is essential for re-establishing normal protein conformation and maintaining cellular homeostasis

433

under various stresses.45 In this study, two HSP70 proteins were upregulated in both the cultivars,

434

with higher levels in Huaguan than in Lvbao (Table S4), partly accounting for the higher DBP

435

tolerance in Huaguan. Small heat shock proteins (smHSPs) are virtually ubiquitous molecular

436

chaperones that can prevent irreversible aggregation of denaturing proteins.46 Increasing data

437

suggest a strong correlation between smHSPs accumulation and plant tolerance to a wide range of


Page 20 of 38

Page 21 of 38


438

environmental stresses.47 Here, two smHSPs, HSP23.6 and HSP17.6, were upregulated in

439

Huaguan with no change in Lvbao, indicating that Huaguan might possess stronger adaptability

440

and tolerance to DBP stress. The DBP-induced upregulation of biotic stress-related proteins in

441

Huaguan, such as disease resistant proteins and chitinases (Table S4), was also seen in aluminum

442

(Al)-tolerant rice cultivars under Al stress.39 The results indicated that Huaguan possessed a better

443

cross-tolerance mechanism to cope with DBP stress compared to Lvbao. The other stress-related

444

DRPs with different abundance patterns in the two cultivars that might mediate the response to

445

DBP stress were likely conducive to the adaptation of B. parachinensis to mediate DBP stress.

446

Overall, when exposed to toxic DBP, and compared to the low-DBP cultivar, the high-DBP

447

cultivar could reprogram protein translation to improve survival when faced with greater pollution

448

stress.

449

3.8. DRPs involved in cell transport and signal transduction related to DBP

450

accumulation. Two kinds of proteins involved in cell transport were detected in this study: Ran

451

proteins and non-specific lipid transfer proteins (nsLTPs) (Table S4). Ran is a small nuclear GTP

452

binding protein that regulates nuclear protein transport and cell cycle progression.48 In this study,

453

three Ran proteins were downregulated in Huaguan with little change in Lvbao under DBP stress,

454

suggesting that Huaguan could adapt better to DBP stress by suppressing the expression of Ran

455

proteins. A similar result has been reported in another study, which suggested that two Ran

456

proteins were suppressed in the more salt-tolerant cucumber cultivar.25 nsLTPs that have a broad

457

lipid-binding specificity in vitro, including to fatty acids, fatty acyl-CoA, phospholipids,

458

glycolipids, and hydroxylated fatty acids, are responsible for the shuttling of lipids between cell

459

membranes and the intracellular transport of lipids. As a typical lipid compound, DBP might be

460

transported by nsLTPs in plant cells. In fact, this study found that nsLTPs in B. parachinensis



461

could combine with DBP by docking simulation and fluorescence quenching experiments (data

462

not shown), and further insights into the precise mechanisms involved in DBP transport in vivo

463

are urgently needed. Thus, the upregulation of nsLTPs in Huaguan might be conducive to DBP

464

transport and accumulation in mesophyll cells.

465

Perception and transmission of stress signals are the central pieces of the plant response to

466

environmental stresses.39 Some DRPs involved in signal transduction in response to DBP stress

467

were identified in this study, including proteins related to the abscisic acid (ABA), jasmonic acid

468

(JA), and mitogen-activated protein kinase (MAPK) pathways, as well as 14-3-3 proteins (Table

469

S4 and Figure 6). ABA signaling plays a critical role in the plant response to abiotic stress through

470

induced antioxidant protection, and the earliest processes occurring in the ABA signal

471

transduction pathway include the interactions among pyrabactin resistance (PYR)/PYR-like

472

(PYLs), type 2C protein phosphatases (PP2Cs), and Snf1-related protein kinases 2 (SnRK2s).49

473

As reviewed by Lee and Luan,49 the interaction between PP2C and SnRK2 causes inactivation of

474

SnRK2 and suppression of the signaling pathway. The overexpression of PYR/PYL could bind

475

PP2Cs to break the inhibition of SnRK2s by PP2Cs, leading to activation of SnRKs and

476

subsequently this could activate the downstream transcription factor__ABRE binding factor

477

(ABF).50 In this study, PYR/PYLs and SnRK2s were upregulated but PP2Cs were downregulated

478

in Lvbao, suggesting that the ABA signaling pathway was activated. By contrast, no regulation of

479

DRPs involved in this signaling pathway was observed in Huaguan, except for an upregulated

480

PP2C. This indicated that the ABA-induced antioxidant pathway in the low-DBP cultivar played a

481

genotype-specific role in coping with DBP accumulation. Similar results were found in the

482

low-Cd genotype of pakchoi in response to Cd stress.10 Jasmonic acid (JA) not only influences

483

numerous developmental processes as a growth regulator in plants, but also acts as an important


Page 22 of 38

Page 23 of 38


484

signal molecule in plant defense against stress.51 As shown in Figure 6, DBP stress stimulated the

485

JA signal transduction network in both cultivars. Three DRPs involved in the JA signaling

486

pathway were upregulated in Huaguan, thus contributing to its higher DBP tolerance, while two

487

proteins related to signal transduction were downregulated in Lvbao (Table S4). The

488

mitogen-activated protein kinase (MAPK) family is believed to play various roles in intracellular

489

and extracellular signaling transduction in plants, which acts as a point of convergence in abiotic

490

stress signaling.52 We found that all of the DRPs identified from the MAPK pathway in Huaguan

491

or Lvbao were upregulated in response to DBP treatment (Table S4), demonstrating that this

492

signaling pathway was activated in both cultivars to adapt to DBP stress. As reviewed by

493

Zelicourt et al.,53 H2O2 production can activate MAPKs as well as ABA and JA, which in turn

494

induce the expression and activities of antioxidant enzymes. In this study, the upregulation of the

495

proteins oxidative stress inducible 1 (OXI1) and nucleoside diphosphate kinase 2 (NDPK2)

496

induced by H2O2 were more pronounced in Huaguan than in Lvbao, resulting in greater

497

antioxidant activity in the former. 14-3-3 proteins are reported to regulate the activities of many

498

proteins involved in signal transduction and they play important roles in stress responses in higher

499

plants.39,54 Here, we identified a 14-3-3 protein which was upregulated only in Huaguan,

500

suggesting that it was helpful in dealing with DBP stress by enhancing signal transduction.

501

Overall, the responses of B. parachinensis to DBP toxicity were regulated in multiple signaling

502

pathways, and the differences in the proteomic responses to DBP stress might account for the

503

variation in DBP accumulation between the two cultivars.

504

In conclusion, major proteomic differences were found between the two crop cultivars

505

differing in DBP accumulation. Some candidate proteins identified from cell wall biosynthesis,

506

basic metabolic processes, DBP-stress responses, cell transport, and signal transduction were



507

predicted for DBP tolerance and accumulation, which helped reveal the molecular mechanisms

508

that contribute to the genotype differences in DBP accumulation. The results greatly enrich our

509

knowledge of the genetic basis of low DBP-accumulating crop cultivars. Further functional

510

characterization of these candidates is essential to the development of new breeding programs for

511

low-accumulation crop cultivars based on molecular assistant breeding techniques.

512 513

ASSOCIATED CONTENT

514

Supporting information

515

The attached supporting document contains DBP-tolerance curves of the two cultivars (Figure S1);

516

representative GC/MS chromatograms of the tested DBP (Figure S2); LC-MS/MS data validation

517

(Figure S3); representative LC-MS/MS chromatogram of the proteomes quantification (Figure

518

S4); iTRAQ data statistics (Figure S5); GO analyses of all the identified proteins (Figure S6);

519

qPCR verification (Figure S7); parameters of the DBP-tolerance curves (Table S1); qPCR primers

520

(Table S2); DBP concentrations in shoots of the two cultivars (Table S3); and a list of DPRs in the

521

two cultivars treated with DBP (Table S4).

522 523 524

AUTHOR INFORMATION

525

Corresponding Author

526

*Ce-Hui Mo. E-mail: [email protected]. Tel: +86 2085220564. Fax: +86 2085226615.

527

Notes

528

The authors declare no competing financial interest.

529


Page 24 of 38

Page 25 of 38


530

ACKNOWLEDGEMENTS

531

This work was funded by the National Natural Science Foundation of China (41703085,

532

41573093), the NSFC-Guangdong Joint Fund (U1501233), the General Financial Grant from the

533

China Postdoctoral Science Foundation (2016M602603), the Natural Science Foundation of

534

Guangdong Province (2017A030313230), the Program of the Guangdong Science and

535

Technology Department (2016B020242005, 2015B020235008), the Research Team Project of the

536

Natural Science Foundation of Guangdong Province (2016A030312009), the project on

537

the Integration of Industry, Education and Research of Guangdong Province (2015B090903070),

538

and the Project of the Guangzhou Science and Technology (201704020074).

539 540

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FIGURES Figure 1. (a) Biomass and DBP concentrations in shoots of two B. parachinensis cultivars under DBP stress. The different uppercase (or lowercase) letters indicate significant differences in DBP concentration (or biomass) at p

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