DcC4H and DcPER Are Important in Dynamic Changes of Lignin

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Omics Technologies Applied to Agriculture and Food

DcC4H and DcPER Are Important in Dynamic Changes of Lignin Content in Carrot Roots under Elevated Carbon Dioxide Stress Ya-Hui Wang, Xue-Jun Wu, Sheng Sun, Guo-Ming Xing, Guang-Long Wang, Feng Que, Ahmed Khadr, Kai Feng, Tong Li, Zhi-Sheng Xu, and Ai-Sheng Xiong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02068 • Publication Date (Web): 06 Jul 2018 Downloaded from http://pubs.acs.org on July 8, 2018

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Journal of Agricultural and Food Chemistry

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DcC4H and DcPER Are Important in Dynamic Changes of

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Lignin Content in Carrot Roots under Elevated Carbon

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Dioxide Stress

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† † ‡ ‡ † Ya-Hui Wang , Xue-Jun Wu , Sheng Sun , Guo-Ming Xing , Guang-Long Wang ,

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† † † † † Feng Que , Ahmed Khadr , Kai Feng , Tong Li , Zhi-Sheng Xu , Ai-Sheng

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Xiong

*,†

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†

State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of

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Horticulture, Nanjing Agricultural University, Nanjing, 210095, People’s Republic of

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China

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‡

College of Horticulture, Shanxi Agricultural University, Taigu, 030801, People’s

Republic of China

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* Please address all correspondence to: Xiong A.S. ([email protected])

ACS Paragon Plus Environment


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ABSTRACT: In our study, isobaric tags for relative and absolute quantification

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(iTRAQ) was conducted to determine the significant changed proteins in the fleshy

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roots of carrot under different carbon dioxide (CO2) treatments. A total of 1,523

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proteins were identified, of which 257 were differentially expressed proteins (DEPs).

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On the basis of annotation analysis, the DEPs were identified to be involved in energy

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metabolism, carbohydrate metabolism and some other metabolic processes. DcC4H

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and DcPER, two lignin-related proteins, were identified from the DEPs. Under

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elevated CO2 stress, both carrot lignin content and the expression profiles of lignin

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biosynthesis genes changed significantly. The protein-protein interactions among

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lignin-related enzymes proved the importance of DcC4H and DcPER. The results of

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our study provided potential new insights into molecular mechanism of lignin content

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changes in carrot roots under elevated CO2 stress.

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KEYWORDS: carrot, carbon dioxide stress, iTRAQ, lignin, DcC4H, DcPER, gene

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expression


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■ INTRODUCTION

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Carrot (Daucus carota L.), belonging to the Apiaceae, is an important economic

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root vegetable.1-3 Carrot is native to the southwestern of Asia, and Afghanistan is the

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earliest evolution center.4-5 There are many nutrients accumulating in carrot fresh

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taproot, such as carotenoids, anthocyanins, vitamins and lipoids.6-7 As a model plant,

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carrot is conductive to exploring the regulatory mechanism and biosynthesis in root

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organs.8 Lignin is a macromolecular substance next to cellulose in vascular plants. It

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can provide the necessary mechanical strength for plant cells and tissues which

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contributes to the transport of water and nutrients in plants.9 Plant contains a large

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amount of lignin in the xylem of roots. Nevertheless, excess lignin can affect the

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growth and development of root vegetables and decline the quality of roots.10 So for

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root crop carrot, lignin content is an important indicator for judging carrot quality.

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Previous studies showed that many key enzymes played important roles in lignin

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biosynthesis. Cinnamate 4-hydroxylase (C4H) is involved in the initial step of lignin

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biosynthesis pathway.11 By catalytic reaction, C4H can promote the biosynthesis of

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p-coumaric acid.12 C4H expresses widely in various tissues, especially in lignified

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roots and cells.13 Ye indicated that C4H activity was closely related to the lignin

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deposition in xylem and phloem fiber cells of Zinnia elegans.14 Meanwhile, Blount

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and his colleagues also demonstrated the reduced lignin content in transgenic tobacco

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together with the decrease in C4H activity.15 The results were also similar in alfalfa

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and Populus.16-17 Peroxidase (PER) is a type of stress-responsive protein which is

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widely distributed in all kinds of organisms.18 PER functions in numerous



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physiological processes, including lignin formation, disease resistance, hormone

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metabolism and so on.19-20

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Since the industrial revolution, the concentration of carbon dioxide (CO2) in

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atmosphere has been continuously increasing due to the influence of human activities,

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and the growth rate was gradually becoming higher.21 With the environment getting

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worse, the atmospheric CO2 levels may meet more pronounced increase at the end of

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the 21st century.22 By affecting the exchange of gases in plant photosynthesis, CO2

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leads to changes in morphological and anatomical structures of plants.23 Meanwhile,

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as a major greenhouse gas, CO2 can also influence the growth and development of

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plants by affecting the temperature of the environment. Currently, many studies have

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been devoted to exploring the mechanism of plants responding to changes of CO2

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levels, which have laid a theoretical foundation for the practice of plant growth and

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development.24-25

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With the development of proteome research techniques, the exploration process

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of linked genes and proteins in response to elevated CO2 of higher plants continued to

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deepen.26-28 Previous research found that a total of 57 differentially expressed proteins

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(DEPs) have been identified in the leaves of rice under elevated CO2 compared with

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the control group. These DEPs were mainly involved in photosynthesis, carbon cycle

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metabolism and energy metabolism pathways.29 After high elevated CO2 stress

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treatment for 12 h, 278 and 440 DEPs were selected in the leaves and roots of

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three-leaf soybean, respectively, of which 50 DEPs were simultaneously present in

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leaf and root tissues.30 However, the research of DEPs related to dynamic changes of


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lignin content in carrot roots is limited due to the lack of proteome information related

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to lignin biosynthesis in carrot.

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In our study, iTRAQ was utilized to investigate the proteomes of carrot fleshy roots

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under the treatments of control CO2 and elevated CO2. DEPs associated with elevated

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CO2 stress were detected. Among these DEPs, DcC4H and DcPER involved in lignin

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biosynthesis pathway were identified. The protein-protein interaction between

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lignin-related enzymes were explored. The lignin content and expression profiles of

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related genes were also detected and analyzed. Our results provided potential insights

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into the molecular mechanism for dynamic changes of lignin content in carrot roots

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subjected to elevated CO2 stress.

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■ MATERIALS AND METHODS

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Plant material and CO2 treatments. Carrot cultivar ‘Kurodagosun’ was selected

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as material during this study. ‘Kurodagosun’ is a stably produced carrot cultivar with

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an orange root. It has been widely applied to molecular research.5, 25 Carrots were

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grown in a climate-controlled growth chamber of the State Key Laboratory of Crop

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Genetics and Germplasm Enhancement, Nanjing Agricultural University (Nanjing,

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China). The growth conditions were set as follows: a photoperiod of 16 h light (25 oC)

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with a light intensity of 300 µmol·m-2·s-1 and 8 h dark (18 oC), 65 % ± 5 % relative

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humidity. Carrots were grown in normal growth environment (~400 µmol·mol-1 CO2

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concentration) for 38 d, then divided into two groups for CO2 treatment. In some



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plants, 3000 and 4000 µmol·mol-1 have been used as concentrations for elevated CO2

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treatment.25 Here, 3000 µmol·mol-1 was set as the elevated CO2 concentration for

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treatment, and atmospheric CO2 concentration (400 µmol·mol-1) was chosen as

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control. The other conditions were the same and remained unchanged. After treatment

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for 30 d, the carrot fleshy root samples were collected and frozen in liquid nitrogen

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immediately, then stored at -80 oC for subsequent experiments. Three independent

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biological replicates were performed for the protein and RNA extraction.

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Protein extraction, digestion, and iTRAQ labeling. The carrot frozen samples

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were ground into powder in liquid nitrogen for protein extraction with NitroExtraTM

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(Cat. PEX-001-250ML, N-Cell Technology). The cell debris was separated by super

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high speed centrifugation at 20,000 rpm for 2 h at 10 oC. Then proteins were

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precipitated with triple volume of cold acetone at -20 oC overnight. After air dried,

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precipitated proteins were washed twice by cold acetone then re-suspended in 8 M

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urea immediately. After chemically reduced by 20 mM DTT in 60 oC for 1 h, the

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samples were alkylated quenched with 40 mM IAA and 10 mM DTT at room

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temperature for 30 min in the dark. Protein concentration of the samples were

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determined using the Lowry’s Assay (DC Assay Kit, Cat. 500-0111, BioRad), and the

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standard of calibration curve was constructed by bovine serum albumin to perform

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quantitative protein assays. Then, a 100 µg peptide mixture was labeled with iTRAQ

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Reagent-8-plex Multiplex Kit according to the manufacturer’s protocol. Two samples

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were labeled with 113 (control) and 114 (treatment), respectively. In short, appropriate

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amount of isopropanol was added to each iTRAQ reagent tube, the contents of each


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vial were transferred to individual sample tubes, their pH was adjusted by 1 M TEAB

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to 7-10. Then the labeling tryptic peptides were incubated at room temperature for 2 h.

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All the labeled samples were dried in spin for subsequent C18 desalting.

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Strong cation exchange (SCX) fractionation. After the labeling, the peptides

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were diluted at a flow rate of 1 mL·min-1 with Buffer A (10 mM KH2PO3, 20 % ACN,

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pH=2.7) and Buffer B (10 mM KH2PO3, 20 % ACN, 0.6 M KCl, pH=2.7), which

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were equipped with PolySULFOETHYLATM (200x4.6-mm, 200A) columns. The

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gradient was set as follows: 0-10 min, 0 % Buffer B; 11-30 min, 15 % Buffer B;

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31-45 min, 45 % Buffer B, 46-53 min, 100 % Buffer B. The fractions were collected

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every minute then further gathered into 12 fractions, followed by dried in spin

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vacuum for liquid chromatography electrospray ionisation tandem mass spectrometry

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(LC-MS/MS) analysis.

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LC-MS/MS analysis. Peptides of the SCX fractions were dissolved in 0.1 % FA

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and then loaded onto a reverse phase C18 column (Chrom XPnanoLC column 75 µm

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id x 15 cm, ChromXP C18 3 µm 120Å) with a flow rate of 300 nL·min-1. A liner

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gradient of acetonitrile (3 %-36 %) in 0.1 % formic acid with a total runtime of 120

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min including mobile phase equilibration was conducted to elute the peptides. The

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samples were analyzed by nano LC-MS/MS using an Eksigent ekspertTM nanoLC 425

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system with AB Sciex TripleTOF® 6600 System. The MS/MS scans were recorded

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with a resolution of ~35,000 full-width half-maximum. The nanospray needle voltage

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was set at 2,300 V. The precursor ions were fragmented in a collision cell using

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nitrogen as the collision gas. Dynamic exclusion was set for 30 s after two repetitive



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occurrences.

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Proteomic database search and quantitative analysis. Raw data files were

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converted to Mascot Generic Format and mzXML format using OpenMS and then

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searched using Mascot Software (Matrix Science). The results were exported in DAT

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files for protein identification. Ratio of each protein was given by geometric mean of

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the protein ratios measured from all replicates. The significance of protein ratios were

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tested by two-tailed student’s t-test. Benjamini-Hochberg multiple hypothesis test

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correction was employed to correct the P-values. The protein ratios of P < 0.05 with

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5 % FDR correction were filtered as differential proteins for following analyses.

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Bioinformatics and annotations. The Gene Ontology (GO) software

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(http://www.geneontology.org) was used to perform the functional category analysis

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according to their molecular function and cellular localization.31 Furthermore, we

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applied Kyoto Encyclopedia of Genes and Genomes (KEGG) databases

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(http://www.kegg.jp/) to execute the pathway analysis.32 To better explore the

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interactions of enzymes related to lignin biosynthesis, we assessed the protein-protein

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interaction network on the base of STRING database.33

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Lignin extraction and determination. The lignin content of the carrot roots were

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extracted and determined based on previous research methods with some

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modifications.34-35 In brief, the carrot root sample was first ground into powder with

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liquid nitrogen and immediately dissolved in 6 mL of 99.7 % ethanol. After

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centrifugation at 14,000 rpm for 20 min, the precipitate was collected and air dried at

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room temperature overnight. Approximately 10 mg of the dried products was


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transferred to a clean tube for follow-up operation. After extraction by 2 M HCl and

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thioglycolic acid and multiple centrifugations, the deposited sediment was dissolved

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in 1 mL of 1 M NaOH for further absorbance measurement at 280 nm. 1 M NaOH

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solution was set as blank control. By using a standard alkali lignin (Sigma-Aldrich), a

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calibration curve was formed for accurate quantification (Figure S1).

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RNA isolation and gene expression assays. Total RNA from fleshy carrot roots

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was extracted by using the RNA simple Total RNA Kit (Tiangen, Beijing, China).

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Then the PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time)

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(Takara, Dalian, China) was utilized to reverse-transcribe RNA into cDNA according

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to the manufacturer’s protocol. Quantitative real-time polymerase chain reaction

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(qRT-PCR) was then performed with SYBR Premix Ex Taq (Tli RNaseH Plus)

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(Takara, Dalian, China) using a CFX96 Real-time System (BioRad, Hercules, CA,

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USA). The reaction condition was set as follows: 95 oC for 30 s, followed by 40

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cycles at 95 oC for 5 s, and 60 oC for 30 s. Each reaction was run in triplicate. The

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gene, DcActin was selected as an internal control.36 2−∆∆Ct method was utilized to

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calculate the relative expression levels of all genes on the basis of threshold cycle.37

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The sequences of all primer pairs for qRT-PCR were designed by Primer Premier 5.0

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software and could be found in Table 1 and Table S1.

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■RESULTS Grow analysis and physiological index determination. To investigate the effect



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of elevated CO2 treatments (3000 µmol·mol-1) on the growth and development of

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carrot roots, we examined the morphological features as well as biomass

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accumulation of the ‘Kurodagosun’ seedlings at 68 days after sowing (DAS). During

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this stage, the carrot roots have undergone the period of enlargement. The fleshy root

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has become orange and swollen. Compared with control, the carrot under elevated

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CO2 treatment had more fibrous root, thus its leave blades and petioles showed

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slightly yellow (Figure 1). Both shoot and root of the seedlings treated with elevated

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CO2 (3000 µmol·mol-1) showed decreases in weight, accompanied by a decrease of

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root-shoot ratio (Figure 2A, B).

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Identification of DEPs in carrot roots under elevated CO2 treatment. In the

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present study, iTRAQ was performed to analyze the proteome differences between

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carrot fleshy roots under different CO2 treatments. A total of 1,523 proteins were

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identified from the two samples. Up- or down-accumulated proteins were determined

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using the treatment/control ratio, and 257 DEPs were identified. Among the identified

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proteins, 168 (65.37 %) of them were up-regulated proteins, and 89 (34.63 %) were

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down-regulated (Figure 3). According to their geometric ratios normalized by log2

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transform, a heat map was established to determine the expression levels of the DEPs

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under elevated CO2 treatment (Figure S2). As shown in the heat map, most of the

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DEPs were up-regulated. Compared to the down-regulated proteins, their expression

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levels were increased to a higher level.

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Classification of metabolic process-specific proteins. In order to categorize standardized protein functions, all DEPs were searched against the GO database. The


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257 DEPs were categorized into 3 groups (‘biological process’, ‘cellular process’, and

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‘molecular process’) based on the results of GO analysis (Figure 4). According to the

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numbers of DEPs, the most represented GO term was ‘cellular process’. In ‘biological

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progress’, the main functional categories were ‘biological process’, ‘response to

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stress’, ‘anatomical structure development’, ‘biosynthetic process’, ‘cellular amino

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acid metabolic process’, ‘sulfur compound metabolic process’, and ‘secondary

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metabolic process’. From the results of ‘molecular process’, the DEPs were mainly

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distributed in ‘molecular function’, ‘ion binding’, ‘transferase activity’ and

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‘oxidoreductase activity’. Based on KEGG analysis, among the DEPs of carrot roots,

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25.12 % were involved in ‘genetic information processing’; 16.91 % in ‘cellular

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process’; 13.04 % in ‘amino acid metabolism’; 12.56 % in ‘carbohydrate metabolism’

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and 10.14 % in ‘energy metabolism’ (Figure 5).

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Comparative analysis of protein and gene expression profiles. In order to

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explore whether gene expression data would validate the changes in protein

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abundance, nine DEPs were selected for transcriptional analysis. Their protein

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information were listed in Table S2, including protein pI, molecular weight and

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geometric ratio. qRT-PCR results indicated that the expression trends of five selected

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genes were consistent with iTRAQ results, including the genes encoding to

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Cytochrome c1 (DcCYT), Pathogenesis-related protein (DcPRP), Polyphenol oxidase

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I (DcPOI), L-ascorbate oxidase (DcLAO) and Ribulose bisphosphate carboxylase

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(DcRUB) (Figure 6). The other four selected genes encoding to Sucrose synthase

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isoform (DcSSI), ATP synthase (DcATPS), Peroxidase (DcPOD) and Catalase



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isozyme (DcCAT), exhibited inconsistent relationships between the patterns of mRNA

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and protein expression profiles (Figure 6). Transcriptional or post-translational

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modifications might be a reasonable basis for this discrepancy. Among these genes,

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the expression level of DcPRP under elevated CO2 treatment turned out to be 35.57

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times of control group. In contrast, the expression level of DcLAO is only 0.14 times

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as much as control.

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Dynamic changes of lignin content in carrot fleshy roots under elevated CO2

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treatment. By protein annotation, two DEPs (DcC4H and DcPER.) involved in lignin

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biosynthesis pathway were obtained from the proteomics data of carrot fleshy roots

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under elevated CO2 treatment (Table 2). Both of the two proteins were up-regulated in

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carrot fleshy roots subjected to elevated CO2 treatment. C4H participates in the

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beginning of lignin synthesis. PER plays a role at the end of the lignin synthesis.

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To further investigate the influence of CO2 treatments on lignin accumulation, we

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measured the lignin content levels of the carrot roots between control and elevated

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CO2 treatment. The lignin content in carrot roots under elevated CO2 treatment was

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significant higher than that in control (Figure 7).

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Protein-protein interaction among enzymes in the lignin biosynthesis pathway.

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We performed a string network analysis to reveal the protein-protein network of DEPs

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in the lignin biosynthesis (Figure 8). Arabidopsis thaliana was chosen to be the basis

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of the orthologs. The thickness of the lines represents the degree of closeness between

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proteins. DcC4H interacted with six lignin biosynthesis related enzymes, DcCOMT,

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Dc4CL, DcCCoAOMT, DcC3’H, DcHCT and DcPAL, and their interactions were


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extremely close. DcPER2 only interacted with DcLAC1, DcF5H and DcCAD.

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DcCOMT could interact with all other lignin biosynthesis related enzymes except

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DcLAC1, DcLAC2 and Dc4CL. In particular, DcLAC2 did not interact with other

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proteins.

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Expression profile analysis of lignin metabolic pathway related genes. To

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explore the molecular mechanism regulating dynamic changes in lignin contents

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under elevated CO2 treatment, the relative expression levels of lignin-related genes

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were determined in the control and treated carrot fleshy roots (Figure 9). Most of

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these genes were decreased under elevated CO2 treatment. The expression levels of

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genes DcPAL, DcC4H, Dc4CL, DcCAD, DcCCoAOMT, DcPER1, DcLAC1 and

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DcLAC2 were significantly down-regulated. Among these genes, the expression level

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of DcLAC1 under elevated CO2 treatment is only 0.20 times as much as control. In

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contrast, the genes DcCCR, DcF5H, DcCOMT and DcPER2 were highly induced

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under elevated CO2 treatment. Compared to the results of iTRAQ, the expression

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levels of DcC4H and DcPER1 showed inconsistent trends with proteins.

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■ DISCUSSION Proteomic analysis has been applied to a variety of plants38-40, there is still a lack of

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research in carrot. Due to the effect of post-transcriptional regulation, the protein

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expression levels cannot be predicted by the help of quantitative mRNA data.

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Proteomic analysis is important in helping us better understand the potential



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molecular mechanism in plants subjected to stress. By iTRAQ, we found that several

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biological processes of carrot were involved in response to elevated CO2 stress

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(Figure 10). Several proteins may cooperate to establish a stress response network in

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carrot under elevated CO2 stress. Among them, we focused on two lignin-related

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proteins. These results provided novel insights into the effects of elevated CO2 stress

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on dynamic changes lignin content in carrot fleshy roots.

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DEPs were involved in multiple metabolic pathways under elevated CO2 stress.

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Elevated CO2 stress affected the carbon fixation in photosynthesis of carrot

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roots. CO2 is an important substrate for photosynthesis in plants. Increasing the CO2

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concentration directly affects the photosynthesis of plants.41 Plants accumulate

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carbohydrates by photosynthesis, which can provide energy for their growth and

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development.42 In this study, we found that both shoot and root weights of the carrot

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under elevated CO2 treatment (3000 µmol·mol-1) were decreased. The results

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indicated that elevated CO2 stress might inhibit photosynthetic efficiency of plants,

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and slow down even stop plant growth. Previous studies showed that several reasons

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led to the decline of photosynthetic capacity after long-term high CO2 concentration

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treatments. Long-term elevated CO2 can reduce the stomatal conductance of plants.43

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In the long term, the content and activity of Ribulose-1, 5-bisphosphate (RUBP) will

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be cut down.23 In addition, the excessive accumulation of carbohydrates can lead to

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feedback effects and further damage the chloroplasts.44 According to the result of

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protein annotation, several DEPs including ATP synthase and ribulose bisphosphate


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carboxylase that play function in carbohydrate metabolism were down-regulated

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under elevated CO2 treatment. These results demonstrated that high concentration

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CO2 could inhibit photosynthesis in plants by restraining the expression of

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photosynthetic proteins and the accumulation of other related components.

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Energy metabolism was regulated to maintain cell homeostasis under elevated

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CO2 stress. Energy metabolism plays important roles in plant development.45 In the

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KEGG pathways, at least 26 and 21 DEPs were involved in ‘carbohydrate metabolism’

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and ‘energy metabolism’, respectively. Most DEPs involved in carbohydrate

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metabolism were up-regulated, including xylose isomerase, sucrose synthase isoform

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and beta-glucosidase. Sucrose synthase is one of the key enzymes that catalyzes

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sucrose into various metabolites in carrot.7 Sucrose accumulation is a protective

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feedback when the plant is subjected to stress, which can provide sufficient carbon

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source for energy consumption.46 The expression level of malate dehydrogenase was

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also up-regulated. The tricarboxylic acid cycle is the most effective way for plants to

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obtain energy by oxidizing sugar or other substances. In the tricarboxylic acid cycle,

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oxaloacetate was produced through the action of malate dehydrogenase.47 Through

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this process, plants complete the catabolism of sugars, lipids and amino acids. At the

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same time, TCA cycle provides precursors for the production of some other

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substances, such as glutamate, aspartic acid and alanine. These observations lead us to

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speculate that plant may increase energy production by glycolysis and tricarboxylic

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acid cycle in order to offset decreased photosynthesis. This adaptive balance

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contributes to normal growth even in the presence of stress.



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Stress-related DEPs were involved in response to elevated CO2 stress. Based on

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the results of iTRAQ, numerous stress-related proteins responded to the high elevated

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CO2 stress. Calmodulin is a second messenger of signal transduction in plant cells,

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and is involved in the response mechanism of abiotic stress.48 By binding with

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EF-hand, Ca2+ sensors can coordinate various signaling pathways and regulate the

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target protein.49 Under CO2 treatment, the expression of EF-hand protein in carrot

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fleshy roots was reduced by 0.4 times, which was consistent with previous studies in

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soybean.50 The results indicated that the expression levels of the proteins related to the

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signal sensing pathway and the transduction pathway changed significantly in the

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early stress response mechanism of plants, these pathways probably played important

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roles in the response mechanism of carrots. Furthermore, kinds of

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pathogenesis-related proteins also responded to the high elevated CO2 stress. However,

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in virtue of different molecular mechanisms in response to stress, some of them were

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down-regulated and others were up-regulated. Expression level of antioxidant

334

enzymes were also up-regulated, such as catalase.

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Dynamic changes of lignin content in carrot roots under elevated CO2 Stress.

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Lignin was involved in response to elevated CO2 stress in carrot roots. Lignin is

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one of the major products of plant phenylpropanoid metabolic pathway.51 It can

338

quickly respond to external environmental stimuli and a variety of biotic or abiotic

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stresses. Water stress can cause a significant increase in lignin content of carrot root.52

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Besides, several studies have reported the relationship between lignin and disease

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resistance.53-54 Saidi and their team found that brittle leaf disease in leaves and roots


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of Phoenic dactylifera resulted in alterations in lignin content.55 Although lignin has

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beneficial functions in plants, excess lignin accumulation is not favorable. Especially

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in carrots, lignin will cause adverse effects on the taste, quality and texture of carrot

345

root.56 In this study, the lignin content of carrot roots has been increased after high

346

elevated CO2 treatment. The result suggested that lignin perhaps participated in the

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response to high concentration CO2 stress in carrot roots.

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DcC4H and DcPER are important in lignin biosynthesis pathway. On the

349

basis of the results of iTRAQ, DcC4H and DcPER were identified as DEPs in lignin

350

metabolism pathway. We did not find differential expression of other key enzymes

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that participated in lignin biosynthesis pathway, indicating the potential vital roles of

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DcC4H and DcPER in the lignin metabolism pathway. After the deamination of

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phenylalanine by PAL, C4H begins to play a role in converting cinnamate

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4-hydroxylase to p-coumaric acid.56 While, PER catalyzes the production of lignin in

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the final step of the synthesis pathway.57 These two key enzymes directly regulate the

356

lignin content in plants. In this study, both expression profiles of DcC4H and DcPER

357

proteins were up-regulated by elevated CO2. The results manifested that DcC4H and

358

DcPER could play important roles in dynamic changes of lignin content in carrot

359

fleshy roots under CO2 treatment.

360

According to the protein-protein prediction, the interactions of lignin related

361

enzymes were predicted. C4H interacted closely with other proteins in lignin

362

biosynthesis pathway. Previous studies have proved that there was a direct

363

relationship between PAL and C4H activity in plants by transgene expression.15



364

However, DcPER2 only interacted with three enzymes in lignin biosynthesis pathway.

365

We deduced that this might relate to the functional and structure diversity of

366

peroxidase in plants. Peroxidase is widely distributed in various plant tissues and

367

many physiological processes.58 The specific action mechanism of different

368

peroxidase enzymes remains to be explored.

369

Impact of elevated CO2 on expression profiles of genes in lignin biosynthesis

370

pathway. In this study, the expression levels of 14 genes in lignin metabolism

371

pathway were detected by qRT-PCR. Most of them were down-regulated under

372

elevated CO2 stress. Only four selected genes DcCCR, DcF5H, DcCOMT and

373

DcPER2 showed significantly increasing trends. The results demonstrated that the

374

lignin content was the integrated result of multistep regulation. In addition, the

375

expression level of DcPER1 was down-regulated which showed the opposite trend of

376

DcPER2. Previous research showed that PER could regulate the formation of lignin

377

with different structures.11 We inferred that different PER genes might perform

378

distinct functions in the lignin biosynthesis pathway. Moreover, we noticed that

379

DcC4H and DcPER expressed inconsistent trends between the patterns of mRNA and

380

protein. The up-regulation of these two lignin related proteins meant that they might

381

play roles in the regulation of lignin content after translation. Meanwhile, elevated

382

CO2 probably also inhibited the transcription of lignin synthesis associated proteins.

383 384 385

In this study, we investigated the effect of elevated CO2 (3000 µmol·mol-1) treatment for 30 d on the roots of carrot cultivar ‘Kurodagosun’. A total of 257 DEPs


Page 18 of 41

Page 19 of 41


386

were identified from the proteomes of carrot fleshy roots using iTRAQ. By GO and

387

KEGG enrichment analysis, we concluded that the ‘carbohydrate metabolism’ and

388

‘energy metabolism’ were involved in the response mechanism of carrots to high

389

concentration CO2 stress. DcC4H and DcPER in lignin biosynthesis pathway were

390

identified from the DEPs. Protein-protein interaction predicted their important roles in

391

lignin metabolism. Different expression levels of lignin related genes were also

392

explored. Our data provides new information that can assist in understanding of the

393

dynamic changes of lignin content in carrot roots in response to elevated CO2 stress.

394



Page 20 of 41

395

■ABBREVIATIONS

396

4CL: 4-coumarate-CoA ligase

397

ACN: acetonitrile

398

C3’H: p-coumaroyl shikimate/quinate 3’-hydroxylase

399

C4H: cinnamate 4-hydroxylase

400

CAD: cinnamyl alcohol dehydrogenase

401

CCoAOMT: caffeoyl-CoA O-methyltransferase

402

CCR: cinnamoyl-CoA reductase

403

CO2: carbon dioxide

404

COMT: caffeic acid O-methyltransferase

405

DAS: days after sowing

406

DEPs: differentially expressed proteins

407

DTT: DL-Dithiothreitol

408

F5H: ferulate 5-hydroxylase

409

GO: Gene Ontology

410

HCT: hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase

411

iTRAQ: isobaric tags for relative and absolute quantification

412

KEGG: Kyoto Encyclopedia of Genes and Genomes

413

LAC: laccase

414

LC-MS/MS:

415

spectrometry

416

PAL: phenylalanine ammonia lyase

liquid

chromatography

electrospray


ionisation

tandem

mass

Page 21 of 41


417

PER: peroxidase

418

qRT-PCR: quantitative real-time polymerase chain reaction

419

SCX: strong cation exchange

420 421 422

■AUTHOR INFORMATION

423

Corresponding Author

424

*Telephone: +86-25-8439-6790; Fax: +86-25-8439-6790.

425

E-mail: [email protected] (A.S.X.)

426

Funding

427

The research was supported by Shanxi Province Coal Based Key Scientific and

428

Technological Project (FT201402-07), Jiangsu Natural Science Foundation

429

(BK20130027, BK20170460), the New Century Excellent Talents in University

430

(NCET-11-0670) and Priority Academic Program Development of Jiangsu Higher

431

Education Institutions (PAPD).

432

Notes

433

The authors declare no competing financial interest.

434 435

■SUPPORTING INFORMATION DESCRIPTION

436

Supplementary Table 1 Primers of genes corresponded to selected DEPs

437

Supplementary Table 2 Proteins for comparative analysis of protein and mRNA

438

expression profiles



439

Supplementary Figure 1 The standard curve line of lignin content

440

Supplementary Figure 2 Expression levels of DEPs in carrot under elevated CO2

441

treatment

442

(PDF)

443


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444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486

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Pharmacol 2001, 41 (1), 471-505. 49. Popescu, S. C.; Popescu, G. V.; Bachan, S.; Zhang, Z. M.; Seay, M.; Gerstein, M.; Snyder, M.; Dinesh-Kumar, S. P., Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays. Proc Natl Acad Sci U. S. A. 2007, 104 (11), 4730-4735. 50. Ma, H. Y.; Song, L. R.; Shu, Y. J.; Wang, S.; Niu, J.; Wang, Z. K.; Yu, T.; Gu, W. H.; Ma, H., Comparative proteomic analysis of seedling leaves of different salt tolerant soybean genotypes. J Proteomics 2012, 75 (5), 1529-1546. 51. Rogers, L. A.; Campbell, M. M., The genetic control of lignin deposition during plant growth and development. New Phytol 2004, 164 (1), 17-30. 52. Becerra-Moreno, A.; Redondo-Gil, M.; Benavides, J.; Nair, V.; Cisneros-Zevallos, L.; Jacobo-Velazquez, D. A., Combined effect of water loss and wounding stress on gene activation of metabolic pathways associated with phenolic biosynthesis in carrot. Front Plant Sci 2015, 6, 837. 53. Kumar, A. K.; Parikh, B. S.; Pravakar, M., Natural deep eutectic solvent mediated pretreatment of rice straw: bioanalytical characterization of lignin extract and enzymatic hydrolysis of pretreated biomass residue. Environ Sci Pollut Res 2016, 23 (10), 9265-9275. 54. Tronchet, M.; Balague, C.; Kroj, T.; Jouanin, L.; Roby, D., Cinnamyl alcohol dehydrogenases-C and D, key enzymes in lignin biosynthesis, play an essential role in disease resistance in Arabidopsis. Mol Plant Pathol 2010, 11 (1), 83-92. 55. Saidi, M. N.; Bouaziz, D.; Hammami, I.; Namsi, A.; Drira, N.; Gargouri-Bouzid, R., Alterations in lignin content and phenylpropanoids pathway in date palm (Phoenix dactylifera L.) tissues affected by brittle leaf disease. Plant Sci 2013, 211 (3), 8-16. 56. Que, F.; Wang, G. L.; Feng, K.; Xu, Z. S.; Wang, F.; Xiong, A. S., Hypoxia enhances lignification and affects the anatomical structure in hydroponic cultivation of carrot taproot. Plant Cell Rep 2018, 37 (7), 1021-1032. 57. Zhao, Q.; Nakashima, J.; Chen, F.; Yin, Y. B.; Fu, C. X.; Yun, J. F.; Shao, H.; Wang, X. Q.; Wang, Z. Y.; Dixon, R. A., LACCASE is necessary and nonredundant with PEROXIDASE for lignin polymerization during vascular development in Arabidopsis. Plant Cell 2013, 25 (10), 3976-3987. 58. Arora, D. S.; Chander, M.; Gill, P. K., Involvement of lignin peroxidase, manganese peroxidase and laccase in degradation and selective ligninolysis of wheat straw. Int Biodeter Biodegr 2002, 50 (2), 115-120.

606


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607

■Legends to Figures

608 609

Figure 1 Morphology of ‘Kurodagosun’ under 400 µmol·mol-1 (Control) and 3000

610

µmol·mol-1 (Treatment) CO2 concentration

611 612

Figure 2 Physiological indexes of carrots under 400 µmol·mol-1 (Control) and 3000

613

µmol·mol-1 (Treatment) CO2 concentration

614

A. Fresh weight changes between carrots under control and treatment

615

B. Root-shoot ratio between carrots under control and treatment

616

“**” indicates a significant difference from the corresponding value of control group

617

at P < 0.01.

618 619

Figure 3 The counts of up- or down-regulated proteins identified under elevated CO2

620

treatments

621 622

Figure 4 GO classification of DEPs identified in CO2 treatments

623 624

Figure 5 KEGG classification of DEPs identified in CO2 treatments

625 626

Figure 6 Comparative analysis of transcription and protein levels in nine DEPs

627

DcCAT, catalase isozyme; DcATPS, ATP synthase; DcPRP, pathogenesis-related

628

protein; DcPOI, polyphenol oxidase I; DcLAO, L-ascorbate oxidase homolog; DcCYT,

629

cytochrome c1-like; DcPOD, peroxidase 12-like; DcSSI, sucrose synthase isoform X1;



630

DcRUB, ribulose bisphosphate carboxylase small chain 1B.

631


632

at P < 0.01.

633 634

Figure 7 Lignin content in carrot roots under different CO2 concentrations

635


636

at P < 0.01.

637 638

Figure 8 Protein-protein interaction analyses among enzymes in the lignin

639

metabolism pathway

640 641

Figure 9 Gene expression profiles of lignin biosynthesis genes in carrot roots treated

642

with elevated CO2

643

“*” indicates a significant difference from the corresponding value of control group at

644

P < 0.05.

645


646

at P < 0.01.

647 648

Figure 10 Simplified diagram of some biological processes under CO2 stress in carrot

649

C4H, cinnamate 4-hydroxylase; PER, peroxidase; RUBP, Ribulose-1, 5-bisphosphate;

650

TCA, tricarboxylic acid cycle

651


Page 28 of 41

Page 29 of 41


Table 1 Primers of genes involved in the lignin biosynthesis pathway

gene symbol

GenBank ID

DcPAL

KU753808

DcC4H

KM359961

Dc4CL

KU757476

DcHCT

KU757477

DcC3’H

KU757478

DcCCoAOMT

KU757480

DcF5H

KX022116

DcCOMT

KX022117

DcCCR

KX022118

DcCAD

KU757483

DcPER1

KU757484

DcPER2

KX022119

DcLAC1

KU757486

DcLAC2

KU757487

primer sequences (forward/reverse) TTGACACATAAGTTGAAGCACCATCC CAATCTGAGGACCAAGCCACTGAG TTGCCTATTCTCGGTATCACAATCG CAGACAATGGTGGAGTGCTTCAA CCAGGCAAGAATTGTTGATCCAGAGT CCCATCACCTACCAATGTTTCAGAAGT AGGTTCAGGTTACTTCCTCCACTCC GATAATCATTGTCCATGCGTGCCAAG AGGTGTGAAGCCAGAGGAAGTTGA TGTCCAGAGGCACTCGCTTGTAT GGCTGATCGGCTACGACAACAC GTAACTCCATCACCAACAGGAAGCAT GATGGCTGTGGCTCACCTTCTG GGACACAACAGGCGTGGAGTT AAGGAGATGCTGTGGTGCTTCA CCGTAATCTGGTATGGCTTCATAACAC TTAGAGGTGAAGGCAGCAGAGAATG AAGAGACTCGTCACTCCAGCAATG AAGAGAATAGGCATCGTTGGATTGG GAAGGAGATGTGCTGATAACAGTGAC TCTGGTGCTCATACAGTTGGTTTCG GTGGTGGTGTCGTTGGCTGATT CACAGACAAGAGGACCAAGCCATAT TCAGTGCCAGTGAGAGGATTGTTC TCTCAACAGAGGACCATCCAATTCATC CCATCCACCAACAGGCACATCTAT ATTCAGAGCCGATAATCCAGGTGTT GGTGGTGGCAGGACAGATTCTTC



Page 30 of 41

Table 2 The annotation and expression of two DEPs involved in lignin biosynthesis pathway

Accession No.

protein symbol

protein description

protein pI

protein molecular total peptides weight

geometric ratio

log2(geometric ratio)

g24733 g3245

DcC4H DcPER

cinnamate 4-hydroxylase Peroxidase

8.99 8.07

58236.85 35266.14

1.7856 1.5044

0.8364 0.5892

1 15


Page 31 of 41


Figure 1 Morphology of ‘Kurodagosun’ under 400 µmol·mol-1 (Control) and 3000 µmol·mol-1 (Treatment) CO2 concentration 980x1006mm (96 x 96 DPI)



Figure 2 Physiological indexes of carrots under 400 µmol·mol-1 (Control) and 3000 µmol·mol-1 (Treatment) CO2 concentration A. Fresh weight changes between carrots under control and treatment B. Root-shoot ratio between carrots under control and treatment “**” indicates a significant difference from the corresponding value of control group at P < 0.01.

598x348mm (100 x 100 DPI)


Page 32 of 41

Page 33 of 41


Figure 3 The counts of up- or down-regulated proteins identified under elevated CO2 treatments 372x239mm (96 x 96 DPI)



Figure 4 GO classification of DEPs identified in CO2 treatments 290x395mm (96 x 96 DPI)


Page 34 of 41

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Figure 5 KEGG classification of DEPs identified in CO2 treatments 556x313mm (96 x 96 DPI)



Figure 6 Comparative analysis of transcription and protein levels in nine DEPs DcCAT, catalase isozyme; DcATPS, ATP synthase; DcPRP, pathogenesis-related protein; DcPOI, polyphenol oxidase I; DcLAO, L-ascorbate oxidase homolog; DcCYT, cytochrome c1-like; DcPOD, peroxidase 12-like; DcSSI, sucrose synthase isoform X1; DcRUB, ribulose bisphosphate carboxylase small chain 1B. “**” indicates a significant difference from the corresponding value of control group at P < 0.01.

1475x1529mm (99 x 99 DPI)


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Figure 7 Lignin content in carrot roots under different CO2 concentrations “**” indicates a significant difference from the corresponding value of control group at P < 0.01.

427x550mm (96 x 96 DPI)



Figure 8 Protein-protein interaction analyses among enzymes in the lignin metabolism pathway 144x76mm (300 x 300 DPI)


Page 38 of 41

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Figure 9 Gene expression profiles of lignin biosynthesis genes in carrot roots treated with elevated CO2 “*” indicates a significant difference from the corresponding value of control group at P < 0.05. “**” indicates a significant difference from the corresponding value of control group at P < 0.01.

1549x949mm (100 x 100 DPI)



Figure 10 Simplified diagram of some biological processes under CO2 stress in carrot C4H, cinnamate 4-hydroxylase; PER, peroxidase; RUBP, Ribulose-1, 5-bisphosphate; TCA, tricarboxylic acid cycle

329x205mm (300 x 300 DPI)


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Graphic for TOC 47x26mm (300 x 300 DPI)


DcC4H and DcPER Are Important in Dynamic Changes of Lignin

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