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Findings of Changes in Primary Metabolism for the Production of Phenolic Antioxidants in Wounded Carrots Cong Han, Peng Jin, Meilin Li, Lei Wang, and Yonghua Zheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01137 • Publication Date (Web): 31 Jul 2017 Downloaded from http://pubs.acs.org on August 1, 2017

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Journal of Agricultural and Food Chemistry

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Physiological and Transcriptomic Analysis Validates Previous

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Findings of Changes in Primary Metabolism for the

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Production of Phenolic Antioxidants in Wounded Carrots

4

5

† † † ‡ *,† Cong Han, Peng Jin, Meilin Li, Lei Wang, Yonghua Zheng

6 7 †

8 9

10 11

College of Food Science and Technology, Nanjing Agricultural University,

Nanjing, 210095, People’s Republic of China ‡

College of Agriculture, Liaocheng University, Liaocheng, 252000, People’s

Republic of China

12 13 14 15

16 17

*

Corresponding author. Tel.:+86 25 8439 9080; Fax: +86 25 8439 5618

E-mail address: [email protected] (Y. H. Zheng)

18 19 20 21 22

1 ACS Paragon Plus Environment


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ABSTRACT: Wounding induces the accumulation of phenolic compounds in

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carrot. This study uses physiological and transcriptomic analysis to validate

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previous findings relating primary metabolism and secondary metabolites in

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wounded carrots. Our data confirmed that increased wounding intensity

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strengthened the accumulation of phenolics accompanied by enhancing respiration

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and showed the loss of fructose and glucose and the increase of energy status in

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carrots. Besides, transcriptomic evaluation of shredded carrots indicated that the

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respiratory metabolism, sugar metabolism, energy metabolism and phenolics

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biosynthesis related pathways, such as “Citrate cycle (TCA cycle)”, “Oxidative

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phosphorylation” and “Phenylpropanoid biosynthesis”, were activated by wounding.

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Also, the differentially expressed genes (DEGs) involved in the conversion of

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sugars to phenolics were extensively up-regulated after wounding. Thus, the

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physiological and transcriptomic data validate previous findings that wounding

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accelerates the primary metabolisms of carrot including respiratory metabolism,

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sugar metabolism and energy metabolism to meet the demand for the production of

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phenolic antioxidants.

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KEYWORDS: carrot, wounding, phenolic antioxidants, RNA-Seq, primary

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metabolism

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■ INTRODUCTION

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In the last few years, postharvest abiotic stresses have been used as an effective

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strategy for enhancing the health benefits and medical properties of fresh fruit and

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vegetables. Many research reports have shown that wounding can obviously induce

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the production of phenolic compounds in a large variety of horticultural crops, such

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as carrot,1 potato,2 onion3 and pitaya.4 Phenolic compounds, as the most important,

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numerous and ubiquitous groups of compounds in plant kingdom, have been

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considered the major contributors to the total antioxidant capacity of fruit,

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vegetables and grains.5 Wound-induced accumulation of phenolic compounds

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usually occurs concomitantly with enhanced antioxidant activity. In this sense, the

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use of wounding provides us a simple and feasible way to gain more phenolic

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antioxidants in horticultural crops.

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Among these stressed crops, carrot has been used as a model system to study the

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synergetic effects of wounding in combination with other abiotic stresses and the

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potential mechanisms of wound-induced accumulation of phenolic compounds.6–12

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To date, some of the physiological and signaling mechanisms on biosynthesis of

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phenolic compounds have been elucidated by carrot tissue. As interpreted by Reyes

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et al (2007),8 the increase in total soluble phenolic (TSP) content can be attributed

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to the faster phenolic synthesis rate, as compared with its decrease or utilization rate.

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Moreover, reactive oxygen species (ROS) have been demonstrated playing a crucial

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role as signaling molecules on the wound-induced accumulation of phenolic

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compounds in carrot.10,12 In addition, most recent studies have shown that the 3 ACS Paragon Plus Environment


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primary metabolism supports the secondary metabolism in horticultural crops to

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produce secondary metabolites when subjected to wounding stress. It was reported

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that wounding stress could result in the accumulation of shikimic acid and

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L-phenylalanine in carrot, both of which are required as carbon sources for the

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production of phenolic compounds.11,13 In plants, the aromatic amino acids are

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drived from sugars.14 The increase of chlorogenic acid isomers contents in wounded

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potato tubers was associated with a pronounced increase in reducing sugar levels,

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indicating a tight correlation between primary carbohydrate metabolism and

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secondary phenylpropanoid metabolism.15

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In general, the burst in respiration has been recognized as a common

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phenomenon in wounded tissues.16 Respiration of sugars via glycolysis, the

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oxidative pentose phosphate pathway (OPPP), and the tricarboxylic acid (TCA)

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cycle can provide precursors for the synthesis of organic acids, amino acids, and

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many other secondary metabolites, including phenolic compounds.17 Accompanied

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by mitochondrial respiration, energy is released and chiefly kept in terms of ATP.18

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In light of these information we wanted to validate previous results that wounded

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products may accelerate their primary metabolisms, such as the respiratory

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metabolism, the sugar metabolism and the energy metabolism to provide sufficient

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precursors and energy for the production of phenolic compounds, thereby repairing

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and healing the damaged tissues.

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To validate these previous findings, we investigated the TSP content, the

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respiration rate, the soluble sugar concentrations and the energy status of carrot 4 ACS Paragon Plus Environment

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tissues under different wounding intensities. Particularly, transcriptomic analysis of

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shredded carrots at different storage period was performed and differentially

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expressed genes (DEGs) involved in the conversion of sugars to phenolics were

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screened. The information generated by RNA-Seq would be useful since it validates

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previous findings on the molecular mechanisms of wound-induced accumulation of

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phenolic compounds in carrot.

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■ MATERIALS AND METHODS

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Plant Material, Processing and Storage. Carrots (Daucus carota L. cv.

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Sanhongliucun) were obtained from a local wholesale market (Nanjing, China) and

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transported to the laboratory within 1 h. Uniform size, color and unblemished fresh

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tissues were selected, washed, air-dried and conditioned overnight at 20 °C before

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processing.

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In the first experiment, whole carrots were cut into three different cutting styles-

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slices, pies and shreds, respectively. Based on the method described by Surjadinata

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and Cisneros-Zevallos,1 the calculated wounding intensities (surface area/weight)

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were 4.5, 6.2 and 19.1 cm2 g-1, respectively, for slices, pies, and shreds. After

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wounding treatment, all wounded samples were placed in polypropylene containers

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and kept at 20 °C for 2 days. Non-wounded carrots were used as control. The TSP

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content and the antioxidant capacity were determined every 24 h, while the

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respiration rate, the soluble sugar contents and the energy status were measured at 0

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(before wounding), 1, 3, 6, 12, 24, 36 and 48 h. At each sampling time, fresh 5 ACS Paragon Plus Environment


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samples were taken, frozen in liquid nitrogen and kept in polyethylene bags at

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–80 °C.

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In the second experiment, shredded carrots with the same storage condition as

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described in the first experiment were used for the transcript profiling analysis.

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Fresh tissues were sampled individually at the following four stages of storage: (1)

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Before wounding (Control); (2) 6 h after wounding (W6); (3) 24 h after wounding

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(W24); (4) 48 h after wounding (W48). The collected samples from each individual

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were pooled together, frozen immediately in liquid nitrogen and kept in sterile tubes

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at –80 °C for RNA separation.

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Total Soluble Phenolics (TSP) Quantification. Quantification of TSP was

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conducted as earlier described by Slinkard and Slingleton.19 Frozen tissues (5 g)

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were extracted with cold methanol (25 mL). The tissue homogenate was preserved

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in covered plastic tubes at 4 °C for 12 h, and centrifuged at 13,000g for 20 min. The

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reaction mixture consisted of 500 µL clear supernatant, 1.5 mL of distilled water

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and 1 mL of Folin−Ciocalteu reagent. The reaction was started by adding 1 mL of

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7.5% Na2CO3 solution (1 mL). After incubating for 2 h at 25 °C, the absorbance at

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765 nm was measured and the TSP content was expressed as mg gallic acid (GAE)

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kg-1 (fresh weight).

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Antioxidant Capacity Analysis. Antioxidant capacity was determined using

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2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging activity. An aliquot

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of 0.2 ml of the same extracts for the determination of TSP content was added to 2.8

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mL of 0.12 mM DPPH solution (dissolved in ethanol) and kept in the dark at 25 °C. 6 ACS Paragon Plus Environment

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After 30 min, the absorbance was measured at 517 nm (A1) and methanol was used

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as control (A0). The results of DPPH inhibition were calculated with the following

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equation:

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% DPPH inhibition=[(A0−A1)/A0]×100

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Measurement of Respiration Rate. The respiration rate was determined based on

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Gao et al with slight modifications.20 Briefly, about 1 Kg of slices, pies, shreds or

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whole carrot tissues were enclosed independently in a 4 L glass chamber with a

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small hole on the lid. Then, the hole was covered by adhesive tape and the container

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was kept at 20 °C for 1 h. Finally, the respiration rate of carrot tissues was assayed

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as CO2 production by using the gas analyzer (CheckMate II, PBI Dansensor,

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Denmark). The respiration rate was expressed as mg CO2 kg−1 h−1.

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Soluble Sugars Analysis. The extraction of soluble sugars from carrot was

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modified from the experimental data described by Macedo and Peres.21 Frozen

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tissues (2 g) were extracted using of 80% ethanol solution (5 mL). A high

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performance liquid chromatography (HPLC) system (Shimadzu LC-20A, Japan)

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equipped with a quaternary pump system (Shimadzu DGU-20A, Japan), an

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auto-injector (Shimadzu SIL-20A, Japan), an evaporative light scattering detector

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(Shimadzu ELSD-LT II, Japan) and a column heater (Shimadzu CTO-20A, Japan)

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was used for quantification. The column used to separate the soluble sugars was a

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4.6 × 250 mm, 5 µm, hydrophilic interaction chromatography (HILIC) column

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(Shodex Asahipak NH2P-50 4E, Japan), which was maintained at 40 °C. Elution

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was performed using the mobile phase of acetonitrile (A) and water (B). The soluble 7 ACS Paragon Plus Environment


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sugar content was identified according to the pure standards and quantified using

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standard calibration curves.

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ATP, ADP, and AMP Contents and Energy Charge Assays. Frozen tissues (2 g)

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were finely ground in perchloric acid (5 mL). ATP, ADP, and AMP were quantified

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using a HPLC system (Agilent 1100) based on a method previously reported by Jin

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et al.22 Energy charge was calculated with the following equation:

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[ATP + 1/2ADP]/ [ATP + ADP + AMP].

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RNA-sequencing (RNA-seq). To obtain the transcriptome of the aforementioned

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four groups, RNAs were isolated from frozen tissue samples using Plant Total RNA

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Extraction Kit (TaKaRa 9769, Shanghai, China). For each group, we performed two

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replications. Sequencing libraries were generated following manufacturer’s

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recommendations (NEB, USA), which were named Control, W6, W24 and W48,

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respectively. After cluster generation, the library preparations were sequenced using

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Illumina HiseqTM 4000. After preprocessing, the data were in a cleaned and

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polished form. Clean data with high quality were used for the subsequent analyses.

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Clean

reads

were

mapped

to

the

reference

genome

of

carrot

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(https://www.ncbi.nlm.nih.gov/genome/?term=Daucus+carota) using Tophat2 (v

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2.1.0)23. The reads numbers mapped to each gene were counted using HTSeq (v

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0.6.0).24 FPKM (reads per kilobase per million reads) were used to quantify the

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mapped whole gene expression levels. Genes with an adjusted P-value 99.9

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(Q30), and the summary of RNA-seq data is shown in Supplementary Table S2.

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Read count data were used to identify the DEGs among samples by using DESeq R

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packages.26 As shown in the Venn diagrams (Supplementary Figure S1), altogether

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13407, 14448, and 14509 DEGs were identified between W6 and Control, W24 and

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Control, and W48 and Control, respectively. There were 4865 up-regulated and

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4837 down-regulated DEGs that were commonly shared among the three pairwise

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comparisons.

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KEGG Analysis of DEGs. To further look into the biological functions of genes in

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carrot, the DEGs were searched against the reference canonical pathways in KEGG.

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This study focused mainly on these KEGG pathways related to respiratory

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metabolism,

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metabolism. Here, a total of 11 pathways were listed in Table 1. It was found that

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wounding resulted in extensive up-regulation of DEGs assigned to the pathways of

sugar

metabolism,

energy

metabolism


and

phenylpropanoid


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“Pyruvate metabolism”, “Pentose phosphate pathway”, “Citrate cycle (TCA cycle)”

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and “Oxidative phosphorylation”, especially in W24 and W48. As these pathways

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are known closely associated with respiratory metabolism,36 this observation may

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account for the remarkable increase in respiration rate observed through time after

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wounding. In general, since metabolism is an integrated network, one pathway may

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have a close link between different metabolic processes. Glycolysis, the oxidization

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of glucose to pyruvate, is relevant to both the respiratory metabolism and the sugar

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metabolism. As shown in the pathway of “Glycolysis / Gluconeogenesis”, the

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number of up-regulated DEGs increased while down-regulated DEGs declined

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through time. Similar variation trend was observed in the pathways of “Fructose

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and mannose metabolism” and “Starch and sucrose metabolism”, indicating an

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enhancement of the degradation of carbohydrates in wounded carrots.

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The metabolic pathways, such as glycolysis, TCA cycle and oxidative

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phosphorylation, are essential for the production of ATP. Pyruvate produced from

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glycolysis is oxidised by the mitochondrial TCA cycle, and electrons from the

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resulting reductant are transferred through the electron transport chain and

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ultimately generating ATP by the oxidative phosphorylation.37 Accordingly, the

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large proportion of up-regulated DEGs, especially those in “Citrate cycle (TCA

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cycle)” and “Oxidative phosphorylation”, may further confirm the elevated

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synthesis of ATP and the increased demand for energy consumption in wounded

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carrots. In addition, approximately 85% of all DGEs belong to the pathway of

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“Phenylpropanoid biosynthesis”, were up-regulated within a short time after 14 ACS Paragon Plus Environment

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wounding. And the pathways of “Biosynthesis of amino acids”, “Phenylalanine,

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tyrosine and tryptophan” and “Phenylalanine metabolism”, which involved in the

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biosynthesis and degradation of some specific amino acids, were also markedly

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stimulated by wounding.

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To visualize the metabolic process in relation to the conversion of sugars to

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phenolics, different KEGG pathways were linked together and depicted in Figure 4.

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The FPKM values of DEGs encoding specific enzymes are shown in Table 2. Our

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data revealed that the expression of genes encoding those sugar catabolic enzymes

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such as invertase, fructokinase and hexokinase seemed not fully stimulated at 6 h,

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but exhibited obvious upward trends at 24 h and 48 h. This observation matched

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with the changes of soluble sugars contents in shredded carrots, indicating the

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growing demands of carbohydrates metabolic intermediates for the biosynthesis of

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amino acids. However, as for the genes in regulating the shikimic acid pathway or

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subsequent conversion of chorismate to phenylalanine, their expressions were

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extensively and dramatically up-regulated at 6 h and kept high levels during

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prolonged storage. This observation conformed previous findings on the variations

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of shikimic acid and L-phenylalanine contents in wounded carrots,11,13 validating

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the coordinated switches in primary metabolism for the production of phenolic

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compounds. Furthermore, the DEGs related with conversion of phenylalanine to

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phenolic profiles was significantly up-regulated after wounding. This result

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confirmed the previous work by Becerra-Moreno et al and Jacobo-Velázquez et

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al,11,12 which validates that wounding has a significant role in activating the 15 ACS Paragon Plus Environment


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expression of genes involved in phenolics biosynthesis, and TSP content was

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increased consequently.

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Validation of Selected DEGs by qRT-PCR Analysis. Phenylalanine ammonium

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lyase (PAL), cinnamate-4-hydroxylase (C4H) and 4-coumarate coenzyme A ligase

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(4CL) are three pivotal upstream enzymes in the phenylpropanoid pathway, which

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dominate phenolics production in plants.38 In this study, 9 DEGs that encode for

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these three enzymes, including three for PAL (LOC108223317, LOC108227736,

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LOC108217754), two for C4H (LOC108223289, LOC108197548), and four for

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4CL (LOC108227923, LOC108193321, LOC108206420, LOC108223931), were

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selected to validate the accuracy and reproducibility of the RNA-Seq data by

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qRT-PCR (Table 3). Generally, except for the DEGs of LOC108227736,

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LOC108197548 and LOC108223931, the other 6 assayed genes were significantly

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up-regulated after wounding and kept high expression levels during storage.

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Correlation between the two methods was measured by scatter plotting log 2 fold

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changes (Figure 5). The expression profiles determined by qRT-PCR are generally

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consistent with the data derived from RNA-Seq, reflecting the reliability of results

347

from RNA-Seq.

348 349

■AUTHOR INFORMATION

350

Corresponding Author

351

*Telephone:

352

[email protected]

+86

25

8439

9080.

Fax:

+86

25


8439

5618.

E-mail:

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Funding

354

This study was financially supported by the National Natural Science Foundation of

355

China (No. 31471632).

356

Notes

357

The authors declare no competing financial interest.

358 359

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D. K. Pulsed electric fields pre-treatment of carrot purees to enhance their

457

polyacetylene and sugar contents. Innov. Food Sci. Emerg. Technol. 2014, 23,

458

79–86.

459

(34) Klotz, K. L.; Finger, F. L.; Anderson, M. D. Wounding increases glycolytic

460

but not soluble sucrolytic activities in stored sugarbeet root. Postharvest Biol.

461

Technol. 2006, 41, 48–55.

462

(35) Yi, C.; Qu, H. X.; Jiang, Y. M.; Shi, J.; Duan, X. W.; Joyce, D. C.; Li, Y. B. 21 ACS Paragon Plus Environment


463

ATP-induced changes in energy status and membrane integrity of harvested litchi

464

fruit and its relation to pathogen resistance. J. Phytopathol. 2008, 156, 365–371.

465 466

(36) Wang, J. Y.; Zhu, S. G.; Xu, C. F. Biochemistry, 3nd ed. Higher Education Press: Beijing, China, 2002, pp. 63–175.

467

(37) Alisdair, R. F.; Fernando, C.; Sweetlove, J. Respiratory metabolism:

468

glycolysis, the TCA cycle and mitochondrial electron transport. Plant Biol. 2004, 7,

469

254–261.

470 471

(38) Weisshaar, B.; Jenkins, G. I. Phenylpropanoid biosynthesis and its regulation. Curr. Opin. Plant Biol. 1998, 1, 251–257.


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Page 23 of 36


472

■Legends to Figures

473

Figure 1. Effect of wounding intensity on TSP content (A) and DPPH inhibition (B)

474

of wounded carrots during 2 days storage at 20 °C. Each data point is expressed as

475

the mean ± SD (n=3). Different letters at each storage time represent significant

476

differences at p < 0.05.

477

Figure 2. Effect of wounding intensity on respiration rate (A), fructose content (B),

478

glucose content (C) and sucrose content (D) of wounded carrots during 2 days

479

storage at 20 °C. Each data point is expressed as the mean ± SD (n=3).

480

Figure 3. Effect of wounding intensity on ATP content (A), ADP content (B), AMP

481

content (C) and energy charge (D) of wounded carrots during 2 days storage at

482

20 °C. Each data point is expressed as the mean ± SD (n=3).

483

Figure 4. Metabolic pathways involved in conversion of sugars to phenolics. FK,

484

fructokinase;

485

glucose-6-phosphate 1-epimerase; G6PI, glucose-6-phosphate isomerase; TK,

486

transketolase; EL, enolase; DAHP, 3-deoxy-D-arabino-heptolosonate-7-phosphate;

487

3DQ, 3-dehydroquinate; 3DD, SD, 3-dehydroquinate dehydratase/shikimate

488

dehydrogenase;

489

synthase; CM, chorismate mutase; ADT, PDT, arogenate dehydratase/prephenate 23

HK,

EPSP,

hexokinase;

A1E,

aldose

1-epimerase;

5-enolpyruvylshikimate-3-phosphate;

ACS Paragon Plus Environment

CS,

G6P1E,

chorismate


AAT,

PAT,

bifunctional

490

dehydratase;

491

glutamate/aspartate-prephenate amino transferase; AA, aspartate aminotransferase;

492

TA, tyrosine aminotransferase; HPT, histidinol-phosphate aminotransferase; PAL,

493

phenylalanine ammonium lyase; C4H, cinnamate-4-hydroxylase; 4CL, 4-coumarate

494

coenzyme A ligase; HCT, shikimate O-hydroxycinnamoyltransferase; C3’M,

495

coumaroylquinate

496

coumaroylshikimic acid; CSE, caffeoylshikimate esterase; COMT, caffeate

497

O-methyl transferase. Dotted line represents that some steps were omitted.

498

Figure 5. Correlation of fold change analyzed by RNA-Seq (x axis) and the data

499

obtained using qRT-PCR (y axis). The values were derived from Table 3.

(coumaroylshikimate)

aspartate

aminotransferase

Page 24 of 36

3'-monooxygenase;

500


and

CSA,

Page 25 of 36


Table 1 KEGG analysis of DEGs related to respiratory metabolism, sugar metabolism, energy metabolism and phenylpropanoid metabolisma W6 vs Control W24 vs Control W48 vs Control KEGG pathways Background genes Up

Down

Up

Down

Up

Down

Pyruvate metabolism

43

37

48

28

54

29

90

Biosynthesis of amino acids

103

67

129

58

117

68

232

Pentose phosphate pathway

18

22

21

16

26

13

46

Citrate cycle (TCA cycle)

35

10

45

5

39

3

54

Glycolysis / Gluconeogenesis Fructose and mannose metabolism

40

53

57

43

62

39

126

10

27

19

23

18

17

50

Phenylalanine, tyrosine and tryptophan biosynthesis Oxidative phosphorylation

27

9

28

7

28

9

54

41

44

67

24

73

18

161

Starch and sucrose metabolism

60

43

76

34

86

34

194

Phenylalanine metabolism

41

16

52

16

54

16

147

Phenylpropanoid biosynthesis

66

13

82

13

85

15

207

a

The number of up- and down-regulated DEGs is determined by comparing with control. Background genes indicate the number of all genes in each pathway.

25



Page 26 of 36

Table 2. Summary of DEGs involved in regulating the conversion of sugars to phenolicsa Description Invertase

HK; hexokinase

FK; fructokinase

A1E; aldose 1-epimerase G6P1E; glucose-6-phosphate 1-epimerase

Gene symbol LOC108218454 LOC108218455 LOC108209529 LOC108222817 LOC108216856 LOC108222818 LOC108206477 LOC108218494 LOC108199548 LOC108209997 LOC108203143 LOC108225510 LOC108199984 LOC108228173 LOC108195110 LOC108197533 LOC108209124 LOC108213261 LOC108220493 LOC108210430 LOC108192895 LOC108215076 LOC108206071

Control 0.00 0.19 0.42 0.06 0.00 0.01 0.00 0.63 71.50 22.91 5.82 0.10 166.89 310.58 9.50 2.29 0.75 0.06 9.07 0.49 25.94 17.96 2.18

26


FPKM value W6 W24 2.43 6.04 23.70 51.88 154.98 272.77 1.03 1.73 0.01 0.01 0.11 0.16 0.46 0.81 5.83 11.78 64.25 58.80 19.14 14.96 8.60 9.86 0.05 0.07 210.12 264.53 186.35 42.39 4.97 1.61 1.39 0.82 6.10 11.57 0.28 0.46 9.09 9.87 1.38 2.27 37.19 53.29 21.67 25.33 1.41 0.31

W48 3.62 70.36 168.60 4.45 1.07 0.76 3.45 24.87 60.06 17.87 9.74 0.46 451.12 52.78 3.50 1.20 28.59 2.38 16.58 4.95 77.27 29.52 0.51

Page 27 of 36


LOC108199656 LOC108196280 G6PI; glucose-6-phosphate isomerase LOC108227561 LOC108214821 TK; transketolase LOC108227965 LOC108209071 EL; enolase LOC108210896 LOC108217564 LOC108192892 LOC108227937 LOC108223971 DAHP synthase; LOC108214600 LOC108223353 3-deoxy-D-arabino-heptolosonate-7-phosphate LOC108218443 synthetase 3DQ synthase; 3-dehydroquinate synthase LOC108228166 3DD, SD; 3-dehydroquinate dehydratase/shikimate LOC108202931 dehydrogenase LOC108204867 LOC108206321 LOC108197089 SK; shikimate kinase LOC108204238 LOC108205161 EPSE synthase; 5-enolpyruvylshikimate-3-phosphate LOC108209808 synthase LOC108209237 CS; chorismate synthase LOC108219299 CM; chorismate mutase LOC108224600 LOC108196652

6.01 6.80 285.61 26.02 23.29 157.20 7.71 6.10 214.94 1664.68 34.20 93.41 2.60 44.70 192.46 0.11 0.25 10.86 4.95 7.19 0.19 5.93 0.35 62.40 4.91 0.98

27


2.80 4.89 185.25 27.04 76.77 178.48 14.20 13.79 151.94 1011.98 29.44 73.75 325.88 480.20 185.59 1.73 0.50 8.17 63.38 26.82 0.19 179.15 1.65 158.43 25.95 22.73

0.47 2.11 98.44 25.13 125.44 226.27 20.70 20.15 89.26 319.27 24.69 50.49 760.89 914.29 188.27 2.84 0.88 3.55 125.44 48.57 0.15 366.28 2.85 267.30 44.09 51.56

0.47 2.65 97.84 32.89 182.91 415.21 26.31 31.18 122.22 430.27 25.15 42.46 793.47 846.12 196.92 9.37 3.44 3.00 119.20 63.98 0.23 437.34 1.89 265.24 29.58 44.51


LOC108210290 ADT, PDT; arogenate dehydratase/prephenate LOC108206009 dehydratase LOC108223049 LOC108205816 LOC108205849 LOC108210565 AAT, PAT; bifunctional aspartate aminotransferase and LOC108204769 glutamate/aspartate-prephenate amino transferase AA; aspartate aminotransferase LOC108205030 LOC108204769 LOC108225092 LOC108214974 LOC108204943 TA; tyrosine aminotransferase LOC108224044 LOC108224339 HPT; histidinol-phosphate aminotransferase LOC108197048 PAL; phenylalanine ammonium lyase LOC108227736 LOC108217754 LOC108223317 C4H; cinnamate-4-hydroxylase LOC108223289 LOC108197548 4CL; 4-coumarate coenzyme A ligase LOC108223931 LOC108227923 LOC108193321 LOC108206420 HCT; shikimate O-hydroxycinnamoyltransferase LOC108223132

Page 28 of 36

6.53 18.92 2.42 3.87 0.20 2.66 5.13

18.19 12.93 5.56 9.42 79.94 208.15 71.56

33.97 6.96 9.32 16.04 173.34 466.99 145.45

37.65 6.95 8.93 15.03 141.92 371.24 216.58

0.01 5.13 1.04 327.40 83.93 0.23 1.42 5.57 1110.10 4.54 0.67 44.14 2.98 6.07 3.97 0.05 0.22 28.22

0.03 71.56 3.80 192.75 64.01 3.57 4.04 8.56 884.30 255.77 143.05 1052.64 2.22 6.48 297.41 19.26 99.63 16.60

0.11 145.45 6.82 73.06 43.71 7.26 6.07 12.82 921.58 540.68 275.75 2196.52 1.21 7.50 617.90 40.21 202.80 1.33

0.25 216.58 12.19 67.69 47.46 5.50 4.84 19.05 781.44 531.85 380.58 1729.01 2.67 10.67 535.07 32.00 145.31 0.67

28


Page 29 of 36


LOC108220879 0.41 28.22 LOC108196831 19.72 335.17 LOC108223171 1.04 34.33 C3'M; coumaroylquinate(coumaroylshikimate) LOC108227431 0.24 21.85 3'-monooxygenase LOC108209804 1.15 141.97 CSE; caffeoylshikimate esterase LOC108224003 53.65 229.35 LOC108196175 0.05 1.44 LOC108194156 0.09 37.07 LOC108227433 14.84 81.64 COMT; caffeate O-methyl transferase LOC108214501 0.46 19.98 LOC108210405 17.03 88.10 LOC108214345 0.11 0.25 LOC108215346 0.00 0.04 LOC108223084 0.47 0.63 LOC108223086 0.00 0.00 LOC108221040 0.70 6.32 LOC108215577 1.00 5.09 a The expression level of each DEG is represented by FPKM value. Control, before wounding; W6, 6 h after wounding; W48, 48 h after wounding.

29


62.70 38.72 663.45 576.58 67.24 146.76 45.05 85.07 282.29 218.27 420.84 296.54 2.52 1.58 77.15 52.79 148.09 136.21 37.77 205.67 177.69 243.40 0.25 0.21 0.04 0.51 0.91 381.91 0.03 28.04 15.84 723.81 9.19 143.40 wounding; W24, 24 h after


Page 30 of 36

Table 3 qRT-PCR verification of 9 DEGs related with the phenylpropanoid biosynthesis in carrota W6 vs Control (log2FC) W24 vs Control (log2FC) Gene description Gene ID

a

W48 vs Control (log2FC)

FPKM

qRT-PCR

FPKM

qRT-PCR

FPKM

qRT-PCR 9.45 -2.25

LOC108223317

phenylalanineammonia-lyase 1

8.43

9.21

9.49

10.33

LOC108227736


―

―

―

―

8.31 -2.58

LOC108217754


6.63

8.78

6.70

8.49

5.66

7.10

LOC108223289

trans-cinnamate 4-monooxygenase

6.83

4.61

6.07

3.27

4.23

LOC108197548

trans-cinnamate 4-monooxygenase-like

5.36 -1.59

1.20

―

―

―

―

LOC108227923

4-coumarate-CoA ligase 1

7.02

7.94

6.68

7.79

5.60

5.71

LOC108193321

4-coumarate-CoA ligase 1-like

9.24

8.39

8.73

7.85

7.70

6.65

LOC108206420

4-coumarate-CoA ligase-like 6

9.54

14.33

8.41

13.43

7.15

11.94

LOC108223931

4-coumarate-CoA ligase-like 7

―

―

1.11

0.47

1.25

-0.16

―, not identified as DEG.

30


Page 31 of 36


■FIGURE GRAPHICS

Figure 1



Figure 2


Page 32 of 36

Page 33 of 36


Figure 3



Figure 4


Page 34 of 36

Page 35 of 36


Figure 5



■TABLE OF CONTENTS GRAPHICS


Page 36 of 36

Physiological and Transcriptomic Analysis Validates Previous

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