Isoproturon-Induced Salicylic Acid Confers Arabidopsis Resistance to

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Agricultural and Environmental Chemistry

Isoproturon-induced salicylic acid confers Arabidopsis resistance to isoproturon phytotoxicity and degradation in plants Feng Fan Lu, Jiang-Yan Xu, Li Ya Ma, Xiang Ning Su, Xin Qiang Wang, and Hong Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04281 • Publication Date (Web): 07 Nov 2018 Downloaded from http://pubs.acs.org on November 29, 2018

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Journal of Agricultural and Food Chemistry

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Title: Isoproturon-induced salicylic acid confers Arabidopsis resistance to isoproturon

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phytotoxicity and degradation in plants

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Running head: Degradation of isoproturon by salicylic acid

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Name of authors: Feng Fan Lua, Jiang Yan Xua, Li Ya Maa, Xiang Ning Sua,b, Xin Qiang

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Wanga, Hong Yanga,b

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Institute: aJiangsu Key Laboratory of Pesticide Science, College of Sciences, Nanjing

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Agricultural University, Nanjing 210095, China;

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Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing Agricultural

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University, Nanjing, China

bKey

Laboratory of Monitoring and

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Mailing address: Weigang No.1, Chemistry Building, College of Sciences, Nanjing

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Agricultural University, Nanjing 210095, China

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*Corresponding author: Hong Yang

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Telephone number: +86-25-84395204

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Email: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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This study identified the effect of salicylic acid on degradation of isoproturon in Arabidopsis.

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Three T-DNA insertion mutant lines pal1-1, pal1-2 and eps1-1 defective in salicylic acid

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synthesis were tested, which showed higher isoproturon accumulation and toxic symptom in the

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mutants. When treated with 5 mg/L salicylic acid, these lines displayed a lower level of

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isoproturon and showed attenuated toxic symptom. RNA-sequencing study identified 2651

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(1421 up and 1230 down) differentially expressed genes (DEGs) in eps1-1 and 2211 (1556 up

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and 655 down) in pal1-2 mutant plants (> 2.0 fold change, p< 0.05). Some of the DEGs covered

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Phase IIII reaction components like glycosyltransferases (GTs) and ATP-binding cassette

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transporters (ABCs). Using ultra performance liquid chromatography-time of fight tandem-mass

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spectrometer/mass spectrometer (UPLC-TOF-MS/MS), thirteen Phase I and four Phase II

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metabolites were characterized. Of these, two metabolites 1-OH-isopropyl-benzene-O-glucoside

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and 4-isopropylphenol-S-2-methylbutanoyl-serine have been identified and reported for the first

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time.

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KEYWORDS: isoproturon; detoxification; degradation; Arabidopsis; salicylic acid

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INTRODUCTION

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Salicylic acid (SA) is one of the plant signal molecules for mediating diverse biological

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processes including defense to environmental stresses.

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salicylic acid is to actively participate in biotic and abiotic stress responses such as systemic

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defense to bacteria or viruses and plant resistance to salt and metal toxicity. 2-5 Recently, several

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studies have demonstrated that pesticide-induced phytotoxicity and cellular damage in plants are

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prevented by exogenous salicylic acid.

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exposed to propazine followed by salicylic acid spraying exhibit improved growth,

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physiological responses and propazine degradation. 9 Salicylic acid also reduces the toxic effect

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of chlorpyrifos (≥ 20 mg kg-1) on wheat by accumulating fewer pesticides in plants.

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Furthermore, degradation of several other types of herbicides in plants and their rhizosphere can

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be intensified by salicylic acid. 7,10,11 The enhanced degradation of isoproturon is associated with

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active glycosyltransferases, suggesting that the process is involved in Phase II mechanism for

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modification and degradation.

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toxicants in the presence of salicylic acid is still poorly understood.

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8,12

6-8

1

One of the major roles played by

Wheat, maize and rapeseed crops growing in soils

10

However, the mechanism underlying the disappearance of

Isoproturon [3-(4-isopropylphenyl)-1,1-dimethylurea] (IPU) belongs to the phenylurea 11,13

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herbicide family widely applied for killing weeds in farmland.

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practical use of isoproturon in the field is beneficial to crop productivity, the long-term input of

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isoproturon into soil has a negative effect on crop production.

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become one of the widespread environmental contaminants because it is frequently traced down

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in soil and ground water.

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prone to enter plants and thus risks the safe crop production.

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way of detoxifying and degrading isoproturon in the crops growing in the herbicide-polluted

16

14,15

Despite of the fact that

In fact, isoproturon has

Isoproturon is hydrophobic, weakly absorbed by soil particles and

3


17,18

It is necessary to find out a


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soils and environment.

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The environmental risk of pesticide residues relies on its dissipation in soils and crops.19,20

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Soils are the major media whereby the pesticides are dominantly degraded. 21 Many efforts have

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been made to remedy pesticide-polluted soil through multiple strategies.

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is the bioremediation by which indigenous microbial communities have been applied to

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degrading the toxic compounds in soil. 23 However, successful elimination of pesticide residues

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requires the long-lasting inoculation of active microbial species into the soil, which makes it

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practically difficult and almost infeasible on a large scale. Plant cultivation in lower

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pesticide-contaminated soil can be practicable because genetic divergence of plant genotypes

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provides valuable sources that can be selectively used for phytoremediation.22,24 In this regard,

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selecting the plants with multiple mechanisms for eliminating pesticides is critically important.9

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These plants bearing such specific traits have much lower levels of pesticide residues. Prior to

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the selection, the molecular understanding of toxicant uptake, root-to-shoot translocation and

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degradation is required for generating genetically engineered plants. 12 We previously found that

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exogenous salicylic acid reduced the herbicide napropamide-induced toxicity in rapeseed

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(Brassica napus).6 Exogenous salicylic acid supply could attenuate the physiological response to

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isoproturon toxicity in wheat

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even rhizosphere as well.

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degradation of isoproturon remains elusive. In this study, we characterized three lines of mutants

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from Arabidopsis exhibiting defective salicylic acid synthesis, and showed weak growth and

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severe injury of the plants under isoproturon exposure. To better understand the degradation

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pathways, comparative analyses of genome-wide transcripts between the mutants and wild-type

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were conducted. Numerous genes encoding enzymes for detoxification and degradation were

11

7

22

The prevailing way

and facilitate degradation of the herbicide in wheat plants and

However, the regulatory mechanism behind the detoxification and

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differentially expressed under isoproturon exposure, suggesting that these potential

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isoproturon-resistant components probably involve the metabolic pathways. Thus, the aim of the

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study is to shed light on how detoxification and catabolism of isoproturon residues are

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intensified by salicylic acid in plants.

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MATERIALS AND METHODS

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Plant growth and treatment. Wild-type seeds of Arabidopsis thaliana (Col-0), the PAL1

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(AT2G37040) and EPS1 (AT5G67160) T-DNA insertion mutants pal1-1 (SALK_022804, Col-0

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background), pal1-2 (SALK_096474, Col-0 background) and eps1-1 (SALK_136105, Col-0

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background) were provided by the Arabidopsis Biological Resource Center. Seeds were

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germinated on the 1/2 Murashige and Skoog (MS) medium (pH 5.7) in a growth chamber at 21

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°C with 260 μmol m-2s-1 photosynthetically active radiation and a 14 h light/10 h dark cycle for

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7 d. 25 Young plants were transferred to 1/2 Hoagland nutrient solution and grew under the same

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condition for 14 d. The wild-type plants along with eps1-1, pal1-2 and pal1-3 were examined in

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the presence and absence of isoproturon. The plants were transferred to the same fresh nutrient

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solution containing 0.2 mg/L isoproturon (98% purity)

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shoots of the plants were sprayed with 5 mg/L salicylic acid (Sinopharm Chemical Reagent Co.)

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once a day until the end of the experiment. The growth and treatment solutions were changed

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every two days.

7

and grew for 6 d. Meanwhile, the

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Transcript analysis. The quantitative reverse transcription polymerase chain reaction

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(qRT-PCR) was used to examine transcripts of PAL1 and EPS1. Twenty day-old plants were

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exposed to isoproturon for 6 d. Total RNA was isolated from tissues. The extracted RNA was

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incubated at 37 C for 30 min with 1 unit of RNase-free DNase I (Takara) and 1 μL 10 reaction 5



12

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buffer.

The RNA quality was examined with agarose gel stained by ethidium bromide and

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checked on for protein contamination (A260 nm/A280 nm ratios). 25 The reverse transcription

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was undertaken based on instruction of a cDNA synthesis kit. The cDNA was kept at -20 C.

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The qRT-PCR was performed based on the following method described previously.

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volume solution contained cDNA, SYBR Premix Ex Taq and primers (Table S1), with the the

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thermal cycling condition: 1 cycle of 95 C for 30 s for denaturation, 40 cycles of 95 C for 5 s

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and 60 C for 34 s for annealing and extension, respectively.

12

A final

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Analysis of dry weight and root growth. Wild-type and mutant plants grew

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hydroponically for 20 d and treated with 0 (control group) or 0.2 mg/L (treatment group)

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isoproturon for 6 d. When harvested, plants were rinsed thoroughly with sterile water. Shoots

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and roots were separately harvested and dried at 105 °C for 20 min and 70 °C for 60 h. The

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dried samples were weighted.

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Chlorophyll quantification and measurement of membrane permeability. Chlorophyll

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of leaves (0.1 g FW) was extracted with 80% (acetone/ultrapure water, v/v) acetone. Total

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Chlorophyll were determined by reading the absorbance at 665 nm and 649 nm, and calculated

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using the formula: chlorophyll content (mg/g FW)=(6.10×OD665+20.04×OD649) × 5/0.1. 26,27

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For conductivity, fresh plant tissues were submerged deionized water and stood for 2 h.

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The conductivity of the sample medium (EC1) was determined by an electrical conductivity

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meter. Samples were heated to 100 °C and kept for 20 min. The conductivity of the killed tissue

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extracts (EC2) was determined. 12 The electrolyte leakage (EL) was expressed with the formula

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EL = EC1/EC2100. 8

137 138

Isoproturon determination in plants. Isoproturon was measured by the method described previously.

7,11

Arabidopsis seedlings grew in 1/2 strength Hoagland nutrient solutions with 0 6


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(control) and 0.2 mg/L isoproturon for 6 d. Fresh tissues were ground and extracted

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ultrasonically three times in 15 mL of acetone–water (3:1, v/v) for 30 min, followed by

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centrifugation at 4000 × g for 10 min. The supernatant was concentrated to remove acetone in a

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vacuum rotary evaporator at 40 C. The residual solution was loaded onto an LC-C18 column

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(Supelco Co. Ltd. USA). Eluent was removed. The column was washed with 4 mL methanol.

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The washing solution was analyzed with high performance liquid chromatography.

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Characterization of isoproturon-degraded products in Arabidopsis. The isoproturon

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metabolic and degraded products were characterized by the previous method. 12 LC-MS analysis

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was performed on a Shimadzu LC 20ADXR LC system (Japan) with an AB SCIEX Triple TOF

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5600 mass spectrometer (USA). The injection was volumed with 20 μL. A Poroshell 120 EC-

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C18 column and a gradient system were applied with the mobile phase A (water +0.1% formic

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acid) and B (acetonitrile) at a rate of 0.3 mL/min. The MS was conducted by AB Sciex Triple

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TOF 5600 system with Accelerator TOF Analyzer and electrospray ionization source. TOF-MS

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parameters included ion source gas 1, 65 psi, ion source gas 2, 65 psi, curtain gas 30 psi, source

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temperature 550 °C and ionspray voltage ﬂoating 5500 V. The APCI positive calibration

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solution was used with the AB SCIEX Triple TOF systems on calibration delivery system once

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every 2 samples to ensure a working mass accuracy of 1

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were the baseline for judging the significant difference of gene expression. 28,29

29

The clean reads were produced by removing the raw reads

False discovery rate (FDR) was applied to assessing the p-value in multiple tests

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Gene Ontology analysis. Gene Ontology (GO) category of differentially expressed genes

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with biological functions was applied to the ultra-geometric test using Benjamini-Hochberg

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correction (http://www.geneontology.org/). GO terms with corrected p-value (p ≤ 0.05) were

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considered as significant enrichment for the DEGs relative to the genome background.

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Statistical analysis. Experiments in the study were independently set up in triplicate. Each

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result shown in the figures was the mean of three replicated treatments, and each treatment

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contained at least 18 seedlings. When harvested, the treated samples were randomly selected.

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The significant differences between treatments were statistically assessed by through analyses of

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variance post hoc test (ANOVA, Tukey’s test). All data were analyzed using the statistical

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software package SPSS 22.0. 8


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RESULTS

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Effect of isoproturon on transcripts of salicylic acid synthetic components and

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endogenous salicylic acid production. In higher plants, two pathways of salicylic acid

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biosynthesis have been proposed.

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concerns the plant synthesis of salicylic acid from cinnamate catalyzed by phenylalanine

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ammonia lyase (PAL).32 The other has been identified by genetic studies, showing the salicylic

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acid production from isochorismate synthase (ICS).31 Additionally, two recently identified

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Arabidopsis genes, PBS3 and EPS1 related to pathogenesis and disease resistance, have been

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proposed to involve the synthesis of an important precursor of salicylic acid biosynthesis.

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Since the ICS pathway is the major route for salicylic acid biosynthesis in Arabidopsis, PBS3

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and EPS1 function under the ICS pathway by catalyzing reactions in the conversion of salicylic

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acid from isochorismate. 31

30,31

One of them identified through biochemical approach

31

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To ensure that salicylic acid is actively involved in mediating response to

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isoproturon-induced toxicity in plants, three lines of T-DNA insertion knockout mutant lines

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eps1-1, pal1-2 and pal1-3 defective in synthesis of salicylic acid in Arabidopsis were identified.

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The genomic sequence of Arabidopsis EPS1 contains only an exon with a coding DNA

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sequence of 1669 bp. The mutant eps1-1 was verified by PCR using specific primers and

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inserted with a T-DNA in exon near the 3’-untranslated region (Figure S1), which, as a

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consequence, led to the null expression of EPS1 (Figure S1). The length of Arabidopsis PAL1 is

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3389 bp long, comprising 2 exons interrupted by 3 introns. The knockout mutant lines pal1-2

206

and pal1-3 have a T-DNA insertion in the second intron and exon of PAL1 gene, respectively

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(Figure S1).33 9



208

As showed in Figure 1, compared to the control (-isoproturon), the transcript level of PAL1

209

in plants was significantly enhanced with the application of isoproturon. Treatment with 0.2

210

mg/L isoproturon increased PLA1 transcripts by 5.7 fold compared to the control (Figure 1A).

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The transcription level of EPS1 under the isoproturon exposure showed a moderate increase in

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transcripts at 0.1 and 0.2 mg/L of isoproturon (Figure 1B). The endogenous salicylic acid

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concentration was measured in plants exposed to 0.2 mg/L of isoproturon using electrochemical

214

method.

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4.0 fold compared to the control (Figure 1C). These results suggested that salicylic acid in its

216

synthetic pathways was disturbed by the contamination with isoproturon and most likely

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involved in isoproturon metabolism in plants.

34

Treatment with isoproturon enhanced the salicylic acid concentration by more than

218

Mutation of salicylic acid-synthetic genes led to compromised growth under

219

isoproturon exposure. Under the normal growth condition (-isoproturon) the root elongation,

220

biomass (dry weight) and chlorophyll concentration were similar between the wild-type and

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mutants (Figure 2). To affirm the regulatory role of salicylic acid, the biomarker electrolyte

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leakage indicating the degree of injury by isoproturon was examined. 7 There was no difference

223

between the mutants and wild-type. When plants were exposed to isoproturon, the growth of

224

eps1-1, pal1-2 and pal1-3 mutants was more negatively affected than that of the wild-type

225

(Figure 2A-D), whereas the electrolyte leakage was higher in the mutant lines than in the

226

wild-type (Figure 2E), suggesting that the mutant plants were more sensitive to isoproturon

227

exposure compared to the wild-type control.

228

We then applied the exogenous salicylic acid to the mutants. In the absence of isoproturon,

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no difference of growth and physiological responses with salicylic acid supply was observed

230

between the mutant and wild-type plants (Figure 2). However, compared to the isoproturon 10


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treatment (0.2 mg/L) alone, concomitant supply with 5 mg/L salicylic acid could significantly

232

improve the growth and physiological response under isoproturon stress except the electrolyte

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leakage of pall-2 and pall-3 (Figure 2). For example, the root length in the wild-type, eps1-1,

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pal1-2 and pal1-3 plants with isoproturon and salicylic acid was increased by 8.99%, 19.30%,

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23.58% and 36.85%, respectively as compared to the control (isoproturon) (Figure 2B).

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Salicylic acid reduced isoproturon concentration in plants. Young plants were

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hydroponically grown for 20 d and transferred to the fresh nutrient solution containing 0.2 mg/L

238

isoproturon. The isoproturon treatment lasted for 6 d. During the treatment time, leaves were

239

sprayed with 5 mg/L salicylic acid once a day. Our studies showed that the three mutant lines

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eps1-1, pal1-2 and pal1-3 usually had higher isoproturon concentrations than the wild-type

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(Figure 3A-C). However, the isoproturon concentrations in both wild-type and mutant lines with

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salicylic acid supply were always lower than those without salicylic acid treatment. We then

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determined the IPU resides in the medium of plant growth. The concentrations of isoproturon

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left in the nutrient solution with the growth of mutant lines were slightly but significantly higher

245

than those with the growth of wild-type (Figure 3D). Treatment with salicylic acid led to only a

246

small reduction of isoproturon concentration in the growth medium than non-salicylic acid

247

treatment (Figure 3D).

248

Characterization of metabolites of isoproturon in salicylic acid loss of function

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mutants. To confirm that salicylic acid was able to lower isoproturon concentrations in plants,

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we identified metabolites of isoproturon by UPLC-LTQ-MS/MS. The accurate mass data (< 5

251

parts per million errors) by high resolution MS were applied to confirming elemental formula.11

252

A total of 13 degraded products via Phase I pathway and 4 glycosylated-isoproturon conjugates

253

via Phase II pathway in isoproturon-exposed and/or salicylic acid -treated plants were 11



254

successfully characterized (Table 1).

255

The relative concentrations of isoproturon-degraded products in eps1-1 and pal1-2 mutants

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were generally lower than those of wild-type, although the differences changed considerably

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(Figure 4A). Among these, the concentration of m/z 135, 175, 205a, 237a and 237b were only

258

half or less than that of the wild-type. The isoproturon-degraded product for m/z 205b in the

259

mutants was vanished. For the rest of products, the degradation rate of eps1-1 and pal1-2 was

260

8-50% of the wild-type. A similar comparative analysis was made on accumulation of

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glucosylated isoproturon-derivatives in the mutants and wild-type. The relative intensities of 4

262

adducts of isoproturon were much lower in mutants than those in wild-type (Figure 4B). For

263

example, the isoproturon-conjugates with m/z 357 in eps1-1 and pal1-2 plants were reduced by

264

48.3% and 31.7%, respectively, relative to wild-type (Figure 4B).

265

Thirteen degraded products were characterized according to the accurate MS data and the

266

appropriate fragmentation patterns from MS2 data (Table 1; Figure S2). Isoproturon peaked at

267

15.01 min. The MS2 data of isoproturon with fragment ions were m/z 134, 119, 72. Metabolite

268

#1 (m/z 193), which peaked at 13.38 min, was considered as Monodemethyl-IPU by loss of

269

isopropyl to form fragment ion m/z 150. Metabolite #2 (m/z 237a) peaking at 8.5 min was

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identified as 2-Methylehanoic-IPU due to the loss of hydroxy, leading to formation of fragment

271

ions of m/z 219. Metabolite #3 (m/z 237b) peaking at 12.85 min with elimination of –O-CH3 (32

272

Da) was identified as 2-Methoxyl-IPU according to the fragment ion of m/z 205. From m/z 187

273

and its fragments at m/z 106/77, the new compound Metabolite #4 was confirmed as

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4-(1-hydroxyl -2-methyl-2-propanyl)-N-methylaniline. For metabolite #5 (m/z 205) peaking at

275

6.05 min, the fragment ion of m/z 160 was generated by the loss of N-dimethyl. Thus, it was

276

identified as isopropenyl-IPU. Metabolite #6 (m/z 175), which peaked at 22.23 min, was

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considered as Isopropenyl-demethyl-methyleneimido-IPU by loss of N=CH2 group to form

278

fragment ion m/z 145. Metabolite #7 (m/z 120) peaking at 1.67 min with one main fragment ion

279

m/z 103 generated by the cleavage of amidogen, was confirmed as 4-Vinylanline. Metabolite #8 12


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(m/z 135) peaked at 26.32 min, and its main product ions was m/z 106 with loss of two methyl.

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Metabolite #9 (m/z 152) peaking at 2.26 min with one main fragment ion m/z 93 generated by

282

the cleavage of isopropanol, was confirmed as 1-(4-Aminophenyl) 2-propanol. Metabolite #10

283

(m/z 209) peaked at 4.67 min, the fragment ion of m/z 165 by loss of isopropyl. The loss of one

284

methyl and one hydroxy from m/z 165 formed the fragment ion of m/z 132. Thus, it is

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N-OH-demethyl-IPU. Metabolite #11 (m/z 205) peaking at 6.05 min generated fragment ion m/z

286

163 by removing an isopropyl. Metabolite #12 (m/z 195), which peaked at 12.10 min, was

287

considered as 2-OH-didemethyl-IPU by loss of –NH2 and –NH2–C=O group to form fragment

288

ion m/z 178 and 150. The mass spectrum of metabolite13# peaked at 4.68 min with

289

223 was generated by addition of 17 Da hydroxy group from isoproturon. Therefore, metabolite

290

13# should be hydroxylated isoproturon (2-OH-isopropyl-IPU).

ion of m/z

291

Four conjugates were identified based on the accurate MS data and the fragmentation

292

patterns from MS2 data (Figure S3). The MS2 spectrum generated from conjugate #1 (m/z 357,

293

tR=13.51 min) showed major fragment ions at m/z 179 and m/z 137. Thus, the conjugate was

294

identified as 1/2-OH-didemethyl-IPU-O-glucoside. For Conjugate #3 (m/z 399, tR=5.28 min),

295

m/z

296

4-Isopropylphenol-S-(2-methylbutanoyl) serine. Conjugate #2 (m/z 399) peaking at 15.23 min

297

generated the fragment ion of m/z 340 by loss of acetaldehyde. The loss of a glucose moiety

298

(220 Da) led to the fragment ion m/z 179. The fragment ion m/z 282 was generated by loss of

299

methyl, and the loss of a glucose moiety (179 Da) led to the fragment ion m/z 120. Thus, this

300

conjugate was identified as 2-OH-Cumene-O-glucoside (conjugate #4, m/z 299, tR=10.32 min).

237

was

produced

by

loss

of

propionylserine,

and

it

was

identified

as

301

Genomic RNA-sequencing revealed that expression of many detoxified genes was

302

altered in eps1-1 and pal1-2. Because mutation of EPS1 and PAL1 triggered toxic phenotypes

303

of Arabidopsis under isoproturon stress, we hypothesized that the isoproturon-responsive

304

transcripts would be changed in eps1-1 and pal1-2 plants. To verify it, the genome-wide 13



305

RNA-sequencing was performed with six libraries prepared by wild-type and eps1-1 or pal1-2

306

mutant lines merged with –isoproturon and +isoproturon treatments. Eighteen samples with

307

three biological replicates (6 treatments  3 repeats) were set up. In total, 88.15 Gb clean reads

308

with 12 (43) samples were sequenced from the libraries using the Illumina HiSeq 2500

309

platform. Assessment of the sequenced data revealed that more than 92.9% of each sample had a

310

quality score of Q30 (those with a base quality  30) (Table S2).

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311

We have obtained 20.2-25.1 million clean reads from each of the six libraries following

312

filtering low quality reads (Table S2). The diagram of FPKM density distribution of each sample

313

was plotted, showing that the vast majority of genes was concentrated but only a small part of

314

genes was scattered, suggesting that expression of these genes was drastically changed (Figure

315

S4). By mapping the reads to Arabidopsis genome, we identified 3106 (1414 up and 1692 down)

316

genes in wild-type, 2651 (1421 up and 1230 down) in eps1-1 and 2211 (1556 up and 655 down)

317

in pal1-2 mutants (> 2.0 fold change, p< 0.05) under isoproturon stress, respectively (Figure 5A,

318

B, E, F; Table S3). The number of the DEGs was further presented by plotting Venn diagrams.

319

In total, 520 genes were specifically induced and 984 genes were repressed in wild-type, while

320

527 genes were specifically induced and 522 genes were repressed in eps1-1 mutants (Figure 5A,

321

B). Similarly, 528 genes were specifically induced and 1457 genes were repressed in wild-type,

322

whereas 670 genes were specifically induced and 420 genes were repressed in pal1-2 mutants

323

(Figure 5E, F). Comparative analysis of DEGs between wild-type and eps1-1 or pal1-2 mutant

324

plants revealed that there were 155 genes specifically upregulated and 383 downregulated in

325

wild-type and 386 specifically upregulated genes and 818 repressed genes were found in the

326

eps1-1 mutants under isoproturon stress (Figure 5C, D). In similar, there were 141 genes

327

specifically upregulated and 784 downregulated in wild-type and 1364 specifically upregulated 14


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genes and 640 repressed genes were found in the pal1-2 line under isoproturon stress (Figure 5G,

329

H). The distribution of DEGs abundance in wild-type and mutant lines was confirmed by the

330

color dots (green, down and red, up) (Figure 5I). For example, the dataset of WT/pal1-2 in the

331

presence of isoproturon showed more red dots (DEGs upregulated) than the green dots (DEGs

332

downregulated) (Figure 5K), suggesting that more DEGs in WT tended to be upregulated

333

compared to those in pal1-2. We further randomly selected eight genes for qRT-PCR validation,

334

which showed that expression of all genes could fit in well with those of the DEGs from

335

RNA-Seq (Figure S5).

336

The DEGs from isoproturon-exposed eps1-1 vs WT and pal1-2 vs WT datasets were

337

subjected to Gene Ontology analysis. Based on the specificity, the DEGs were classified into

338

three groups including biological process, cellular component and molecular function (Figure

339

S6). Some categories such as stimulus response, antioxidant activity and protein binding

340

transcription factor activity were identified.

341

Genes involved in isoproturon stress were differentially expressed in mutant lines. We

342

focused on identifying some detoxified-genes from different families encoding cytochromes

343

P450 (CYP), glutathione S-transferases (GST), ATP-binding cassette transporters (ABCs) and

344

glycosyltransferases (GTs) from the datasets of eps1-1 vs WT (+isoproturon) and pal1-2 vs WT

345

(+isoproturon)(Table S4, S5). Among those, 25 P450, 13 GST, 10 ABCs, and 11 GTs were

346

identified from eps1-1 vs WT and 13 P450, 10 GST, 5 ABCs, and 3 GTs were identified from

347

pal1-2 vs WT (Table S4, S5). Expression of all genes was shown to be downregulated under

348

isoproturon exposure, suggesting they were under the direct or indirect control of AtEPS1 and

349

AtPAL1. Despite the fact that both genes AtEPS1 and AtPAL1 control the salicylic acid synthesis

350

and had the similar responses to isoproturon, the isoproturon metabolism guided by AtEPS1 and 15



Page 16 of 33

351

AtPAL1 was found to be different. For example, there were 13 cytochrome genes found to be

352

downregulated in pal1-2 line, whereas there were 25 in eps1-1 line (Table S4, S5).

353 354

DISCUSSION

355

We provided evidence that salicylic acid was able to regulate the isoproturon-induced

356

toxicity in Arabidopsis. We showed that plants sprayed with 5 mg/L salicylic acid in the

357

presence of 0.2 mg/L isoproturon displayed attenuated toxic symptom manifested as enhanced

358

root elongation, total mass accumulation and chlorophyll concentrations. This observation was

359

reinforced by the data of reduced damage of plasma membrane. The salicylic acid-mediated

360

reduction of isoproturon-induced toxicity to plants has been associated with the decreased

361

accumulation of isoproturon in both plants and the media where the plant grew, indicating that

362

salicylic acid can reduce the isoproturon toxicity in the plants.

363

Salicylic acid as a plant signal molecule plays pivotal roles in plant growth, development 5,7,35

364

and resistance to various biotic and environmental stresses.

365

underlying the detoxification and biodegradation of toxic organic compounds is poorly

366

understood. The present studies showed that loss of salicylic acid function mutants led to more

367

sensitivity to isoproturon in the growth of Arabidopsis compared to wild-type (Figure 2).

368

Provision with exogenous salicylic acid could partially rescue the phenotypes in the presence of

369

isoproturon, suggesting that the capability of resistance to isoproturon was impaired to some

370

extent as a result of the salicylic acid disfunction. Consistent with it, mutation of salicylic acid

371

function led to more accumulation of isoproturon in eps 1-1, pal 1-2 and pal 1-3 lines than

372

wild-type, whereas more isoproturon was detected in the mutant growth media than in the

373

wild-type growth media (Figure 3). These results suggested that disruption of salicylic acid 16


However, the mechanism

Page 17 of 33


374

responsive genes would most likely reduce the capability of isoproturon degradation in the

375

plants and uptake of isoproturon from roots. On the other hand, a generally lower level of the

376

isoproturon-degraded products and conjugates was examined in the mutants compared to the

377

wild-type (Figure 4). From these observations we inferred that the capability of isoproturon

378

metabolism or degradation in the mutants would be impaired.

379

To support the assumption and better understand the mechanism leading to salicylic

380

acid-mediated detoxification of isoproturon in plants, we profiled transcriptome from eps1-1 and

381

pal1-2 mutant lines under isoproturon exposure. Substantial numbers of specific genes (522) in

382

eps1-1 and (420) in pal1-2 were found to be repressed, indicating that salicylic acid is necessary

383

for plant response to isoproturon stress (Figure 5). The Gene Ontology analysis revealed that

384

some pathways were related with isoproturon stress response. Of these, genes involved in Phase

385

I, II and III metabolisms should be the point of interests,

386

glycosyltransferases, cytochrome, glutathione S-transferases, and ABC transporters were

387

involved in the isoproturon-induced modification and degradation.

22,36

because genes encoding

388

The Phase I enzymes cytochrome P450s are a group of membrane-integrated enzymes

389

relying on NADPH–P450 oxidoreductase complex. 37,38 The activities of cytochrome P450s are

390

composed of cytochrome P450 protein and NADPH-P450 oxidoreductase to transfer reducing

391

equivalents from NADPH to the cytochrome P450. 37,38 The biochemical process is engaged in a

392

wide range of reactions such as biosynthesis of secondary metabolites, signaling molecules,

393

defense-related chemicals and plant hormones.

394

whose transcripts were substantially increased when plants were exposed to 2,4,6-trinitrotoluene

395

and munition hexahydro-1,3,5-trinitro-1,3,5-triazine in Arabidopsis.

396

one of the important detoxified enzymes in Phase II reaction that catalyze the conjugation of

36

We detected a CYP gene (AT3G28740),

17


39

Glycosyltransferases are


Page 18 of 33

397

toxicants with aglycone like sugar to make them easy for degradation and detoxification.40,41 For

398

example, a glucosyltransferase from Arabidopsis activates the metabolism of the persistent

399

pollutant 3,4-dichloroaniline.

400

herbicide atrazine in rice plants.

401

induced by both salicylic acid and jasmonate when plants were infected by Fusarium

402

pseudograminearum. 43

42

We recently identified a GT that sufficiently degraded a 12

In wheat, a glucosyltransferase gene (CD876318) was

403

We have identified a set of Phase II enzymes (such as GSTs) and ABC transporters from

404

the Arabidopsis. GSTs catalyze the conjugation of toxicants and glutathione (GSH), and the

405

generated GSH S-conjugate is sequestrated into subcellular organelles like vacuoles for further

406

degradation.

407

membrane proteins responsible for uptake, allocation and detoxification of a wide range of

408

metabolites and xenobiotics.

409

major role of this group would help move toxicants to subcellular compartments for degradation.

410

22

411

function roles remain to be elucidated.

24,44

The plant ATP binding cassette (ABC) transporters are one of the integral

45

ABC transporters belong to Phase II reaction components; the

We have identified a total of 15 members relevant to salicylic acid from Arabidopsis but their

412

Based on the identified structure of metabolites, the catabolism and detoxification

413

pathways of isoproturon (IPU) are summarized in Figure 6. Except metabolite 11#, other twelve

414

products

415

hydroxyl-2-methyl-2-

416

isopropenyl-demethyl-methyleneimido-IPU,

417

1-(4-Aminophenyl) 2-propanol, N-OH-demethyl-IPU, 2-methoxyl-IPU and 2-methoxyl-IPU

418

were generated through O-methylated dealkylation, dehydrogenation or hydrolysis reaction by

419

Phase I reaction. We also detected three O-glycosylated and one S-conjugated isoproteron

monodemethyl-IPU,

2-methylehanoic-IPU, propanyl)-N-methylaniline, 4-vinylanline,

18


2-Methoxyl-IPU,

4-(1-

isopropenyl-IPU, desisopropyl-IPU,

Page 19 of 33


420

conjugates involved in Phase II reaction. Our data are potentially useful for enhancing

421

isoproturon degradation in plants growing in isoproturon-contaminated environment and

422

phytoremediation. Further charactering the components would better understand the detailed

423

degradationpathways to the complete disappearance of isoproturon in Arabidopsis.

424 425

ACKNOWLEDGEMENTS

426

The authors acknowledge the financial support of National Natural Science Foundation of China

427

(No. 21577064).

428 429

SUPPORTING INFORMATION AVAILABLE

430

This information is online available.

431

432

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Graphic Abstract

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561 562 563 564 565 566 567 568 569 570 571 572 25



573

574 575 576

Figure 1. Effects of isoproturon (IPU) on transcriptional expression of PAL1 and EPS1 and SA

577

concentration in Arabidopsis. Twenty day-old young plants grew in 1/2 strength Hoagland

578

solution containing IPU at 0-0.8 mg/L for 6 d. qRT-PCR was used to measure expression of

579

PAL1 (A) and EPS1 (B). The concentration of SA in plants was quantified using

580

electrochemical method (C). Vertical bars represent standard deviation of the mean. Data with

581

the different letters indicate the significant difference between the treatments (p < 0.05).

582 583 584 585

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Figure 2. Analyses of growth responses of Arabidopsis wild-type (WT) and three mutants

590

eps1-1, pal1-2 and pal1-3 exhibiting SA defective in synthesis under isoproturon (IPU)

591

exposure. Twenty day-old young plants grown in 1/2 strength Hoagland nutrient solution were

592

exposed to 0 and 0.2 mg/L IPU with or without 5 mg/L SA for 6 days. (A) Images of WT and

593

three mutant seedlings of Arabidopsis. (B) Elongation of Arabidopsis roots. (C) Dry mass of

594

Arabidopsis. (D) Chlorophyll content of Arabidopsis. (E) Membrane permeability of

595

Arabidopsis. Vertical bars represent standard deviation of the mean. Data followed by different

596

letters were significantly different between the treatments (p

Isoproturon-Induced Salicylic Acid Confers Arabidopsis Resistance to

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