Differentially Expressed Protein Are Involved in Dynamic Changes of

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Cite This: J. Agric. Food Chem. 2019, 67, 7547−7560

Differentially Expressed Protein Are Involved in Dynamic Changes of Catechins Contents in Postharvest Tea Leaves under Different Temperatures Rui-Min Teng,† Zhi-Jun Wu,† Hong-Yu Ma,‡ Yong-Xin Wang,† and Jing Zhuang*,† †

Tea Science Research Institute, College of Horticulture and ‡College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China

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ABSTRACT: In this study, isobaric tags for relative and absolute quantitation (iTRAQ) technology were used to investigate three samples from postharvest tea leaves that were treated at room temperature (25 °C, control group), high temperature (38 °C), and low temperature (4 °C) for 4 h. In heat and cold treatments, a total of 635 and 566 differentially expressed proteins (DEPs) were determined, respectively. DEPs were annotated to GO and KEGG databases, which revealed that DEPs involved in various aspects of biological process. Three catechins-related DEPs, CsCHI, CsF3H, and CsANR, were identified. Both catechins contents and the expression profiles of catechins biosynthesis-related genes changed significantly under different temperature treatments. The correlations between catechins contents, gene expression profiles, and DEPs were analyzed. This study provides potential new insights into the molecular basis for tea production of postharvest leaves and catechins content changes at diverse temperature conditions and will guide the improvement of tea-processing technology. KEYWORDS: iTRAQ, catechins, temperature treatment, gene expression, postharvest leaves, tea plant

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INTRODUCTION

On the basis of different processing techniques, the postharvest tea leaves could be processed into various flavors of tea.14,15 Withering is the first procedure for processing white tea, black tea, and oolong tea, etc,16 that is, tea leaves are dehydrated for several hours to promote withering, which changes the appearance and substance of the tea leaves. The main purposes of withering were to improve tea quality by evaporating moisture, enhancing enzyme activity, and adjusting the characteristic ingredients of postharvest tea leaves. Withering is a physiological, biochemical, and molecular regulatory process that affects the final color, taste, and aroma of the tea.16−18 There are many factors that affect the withering process of tea leaves, including withering temperature, time, and light. High or low temperature has different impacts on substance conversion during withering. Tea withering was affected by temperature conditions, which should be controlled within a certain range.16,19 Due to the rapid dehydration of tea leaves under high temperature, withering had to face the challenges of high temperature, especially in summer and autumn. By contrast, tea leaves are dehydrated slowly at low temperature, and the withering of tea leaves also faces the effects of low temperature, especially in spring and winter. Therefore, the technical process of tea leaf processing is always adjusted based on temperature and humidity conditions. Previous studies have demonstrated that the physiological transformations in tea

Tea plant [Camellia sinensis (L.) O. Kuntze], is a leaf-used economic crop. As a nonalcoholic beverage, tea leaves are rich in a variety of ingredients that are beneficial to humans.1,2 These secondary metabolites include mainly catechins, theanine, and polysaccharides. Numerous studies showed that tea plays effective roles in the prevention of cancer, cardiovascular, and neurodegenerative diseases.1−5 The catechins are the main ingredients in tea plant and can reach 12−24% of the weight of dry tea leaves.6 The astringency of tea may involve many compounds, and catechins are the main astringent substances of tea.6,7 Catechins are abundant in tea leaves, including catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), epicatechingallate (ECG), catechingallate (CG), gallocatechin gallate (GCG), and epigallocatechin gallate (EGCG).8 Previous studies revealed that many key enzymes are involved in the biosynthesis of catechins, including chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), and anthocyanin reductase (ANR). Yellow chalcone is catalyzed by CHI to transform into colorless naringenin.9 Naringenin is a direct precursor of all flavonoids synthesis and one of the key genes controlling the content of flavonoids in plants. The synthesis of flavanones was catalyzed by F3H to produce dihydroflavonol.10 These dihydroflavonols are intermediate products of synthesis of flavonols, flavanols, and anthocyanins. In the presence of NADPH, ANR converts reduced anthocyanins into corresponding EC and EGC.11,12 In tea processing, EGCG, ECG, EC, and EGC were isomerized to form corresponding transcatechins GCG, CG, C, and GC, respectively.13 The transcatechins can also be formed in plant biosynthesis.8 © 2019 American Chemical Society

Received: Revised: Accepted: Published: 7547

March 16, 2019 June 4, 2019 June 5, 2019 June 5, 2019 DOI: 10.1021/acs.jafc.9b01705 J. Agric. Food Chem. 2019, 67, 7547−7560

Article

Journal of Agricultural and Food Chemistry

LC-MS/MS Analysis. LC-MS/MS analysis was performed according to our previous report with some modification.29 Briefly, before dissolving in 4 mL of SCX buffer A (25 mM NaH2PO4 in 25% acetonitrile, pH 2.7), the samples were mixed and freeze dried. An Ultremex SCX column (4.6 mm × 250 mm) was used to fractionate peptides by high-pressure liquid chromatography (HPLC, Agilent 1200). The sample was separated by an Exigent Nano LC-Ultra 2D system (AB Sciex). Data acquisition of the mass spectrometer was performed with a Triple TOF 5600 mass spectrometer and a Nano Spray III Source (AB SCIEX). Protein Identification and Quantification. The Protein Pilot Software v4.0 was used to analyze the iTRAQ data through the Uniprot Vitis vinifera database based on the Paragon algorithm.30 According to the PSPEP Software, an automatic decoy database search strategy was used to evaluate the FDR. The distinctive peptides of iTRAQ proteins (FDR < 1%) were regarded for analysis. Identification of DEPs was preformed according to the proportions of differently labeled proteins. The proteins with a fold change more than 1.5 or less than 0.67 and q value less than 0.01 were regarded as DEPs. The q value was used to estimate the FDR and calculated based on the p value, which is a widely used statistical method. Bioinformatics and Annotations. To further functional analysis, all DEPs were searched through the UniProt Arabidopsis thaliana database. Accompanied by a cutoff E value of 1e-10, the best identity of the hit proteins was considered to be homologous. The DAVID toolkit was utilized to analyze the Gene Ontology (GO). The canonical biochemical pathways were analyzed by implementing the KEGG database. According to the STRING database, PPI networks were constructed to better understand the interactions of DEPs.31 Catechins Extraction and Determination. The catechins were extracted according to Liu’s method.6 Briefly, about 0.2 g of dry weight of samples was extracted in 5 mL of methanol/water (7:3) and centrifuged for 10 min at 3500 rpm. The above step was repeated once. Finally, the samples were filtered by a 0.45 μm organic membrane and analyzed with UPLC (Waters, USA). Three technical replicates were performed. Mobile phase A consisted of doubledistilled water and 1% (v/v) methanoic acid. Mobile phase B consisted of double-distilled water, 1% (v/v) methanoic acid, and 80% (v/v) acetonitrile. A Waters ACQUITY UPLC H-class system with a T3 column (2.1 mm × 100 mm, 1.7 μm) was used for chromatographic separation. The other conditions were set to a 0.35 mL/min flow rate, 280 nm detection wavelength, and 30 °C column temperature. The gradient elution procedure is presented in Table 1.

leaves withering processes are affected by different temperatures.16,19 Proteome not only reflects the dynamic changes in the biological processes of gene regulation but also provides biological information on post-translational functions.20,21 Currently, proteomics have been analyzed and identified in multifarious species. A common technique for isolating and identifying proteins is two-dimensional electrophoresis, while it needs to face the challenge of ensuring sensitivity, high resolution, and repeatability.22,23 The isobaric tags for relative and absolute quantitation (iTRAQ) is a method of peptide labeling. This method can precisely quantify the proteins with wider abundance from complicated samples and dispose of test nonrepeatability. 24 Consequently, proteomics based on iTRAQ has been extensively applied in varied species, including microbes and plants.25−27 To date, the metabolism of catechins of postharvest leaves in tea plant (C. sinensis) under temperature treatment is still unclear. “Longjing 43”, an excellent tea plant cultivar widely planted in China, bred from “Longjing” population, is a national wide cultivar with some considerable agronomic traits, including good stress resistance, early germination, and appropriate phenol ammonia content, etc. In this study, the proteomes on postharvest tea leaves of “Longjing43” subjected to three temperature treatments, including room temperature (25 °C, CK), high temperature (38 °C, S1), and low temperature (4 °C, S2), were analyzed by using iTRAQ technology. Biological functions of DEPs were analyzed in heat or cold withering. Three DEPs (CsCHI, CsF3H, and CsANR) that participated in the catechins biosynthesis pathway were identified. The catechins-related enzymes with protein−protein interactions were compared and analyzed. The changes of catechins contents and expression profiles of related genes were also detected under three different temperature treatments. The results will contribute to understand the molecular mechanism of postharvest tea leaves subjected by different temperatures and guide the improvement of tea-processing technology.

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MATERIALS AND METHODS

Plant Material and Withering Treatments. “Longjing43” is a tea plant variety with good characteristics of stress resistance. Oneyear-old asexual propagated seedlings of tea plant were grown in a plug tray and kept in a light incubator in Nanjing Agricultural University. The conditions of the light incubator were set as follows: 16-h light (24 °C)/8-h darkness (20 °C) and 70% relative humidity. About 100 tea seedlings were selected to ensure normal physiological status. The first leaf, second leaf, third leaf, and immature stem were processed with withering treatments at room temperature (25 °C, CK), high temperature (38 °C, S1), and low temperature (4 °C, S2) for 4 h. Normal temperature (25 °C) treatment was selected as the control group. After each treatment the tea leaves were picked, immediately frozen in liquid nitrogen, and then saved at −80 °C for subsequent experiment. Protein Extraction, Digestion, and iTRAQ Labeling. Total protein per sample was disintegrated and dissolved in lysis solution, incubated for 1 h at 30 °C, and then centrifuged for 15 min at 12 000 rpm. According to the Bradford method, protein concentrations were subsequently determined.28 Total protein of 100 μg was dissolved in dissolution buffer for each sample (AB Sciex, Foster City, CA, USA). In accordance with the manufacturer’s manual, the samples were used to reduce, alkylate, and trypsin-digest that labeled subsequently with iTRAQ Reagent-8 plex Multiplex Kit (AB Sciex). Three samples were tagged with iTRAQ corresponding to 113 (CK), 114 (S1), and 115 (S2), respectively.

Table 1. Gradient Elution Profile for UPLC Analysis elution time (min) mobile phase (% (v/v))

0

3

8

8.1

10

A B

95 5

95 5

50 50

95 5

95 5

Gene Expression Assays. RNA sample from the tea leaves was isolated using an RNA Isolation Kit (Huayueyang, Beijing, China), reverse transcribed into cDNA with a PrimeScript RT reagent kit (TaKaRa, Dalian, China). The quantitative real-time PCR (RTqPCR) was completed using SYBR Premix Ex Taq (TaKaRa, Dalian, China); three technical replicates were performed for all reactions. The 2−ΔΔCt method was used to count the relative expression levels of all genes.32 CsTBP gene was applied as internal control gene.33 All primers for RT-qPCR are presented in Tables S1 and S2. Statistical Analysis. The SPSS 17.0 software was used to analyze the data. Duncan’s multiple-range test was used to detect the differences in the expression levels of catechins metabolism-related genes at a P < 0.05 probability level. Data was expressed as the mean ± standard deviation. The correlation between the catechins contents and the expression levels of genes and DEPs was analyzed by Pearson’s method.34 7548

DOI: 10.1021/acs.jafc.9b01705 J. Agric. Food Chem. 2019, 67, 7547−7560

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Figure 1. Counts of DEPs identified from tea leaves under withering treatments. (A) Heat withering (S1/CK). (B) Cold withering (S2/CK).

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irregular, injured, and unsteady proteins.35 A heat shock protein (HSP) was found ranking in the second upregulated protein in heat withering. HSP is easy to be activated by hightemperature stress.36 The lowest expression protein in heat withering was a coatomer subunit epsilon-1 protein, which is related to protein transport.37 Transducin/WD40 domaincontaining protein has the lowest expression in cold withering. Transducin/WD40 domain-containing protein plays key roles in varied biological processes, for instance, cell division, apoptosis, and light signaling.38 We noticed many identical upregulated proteins in both heat and cold withering treatments but downregulated proteins are not. GO Annotation of DEPs Identified in the Heat and Cold Withering Treatments. The whole DEPs were investigated in the GO database in order to classify standardized protein functions. In heat withering, a total of 3282 GO terms were allocated to 634 DEPs. Meanwhile, a total of 3058 GO terms were allocated to 665 DEPs in cold withering. Three GO classes, namely, biological process, cellular component, and molecular function, contain 2266, 335, and 842 terms, respectively. On the basis of P value ranking by ascending order, the top 20 terms of “biological process”, “cell component”, and “molecular function” were calculated (Figure 3). In “biological process”, “organo nitrogen compound metabolic process” and “small molecule metabolic process” terms were comprised of the largest number of DEPs in both heat and cold withering treatments. In contrast, the “cell component” and “molecular function” terms with the largest number of DEPs presented differences between heat and cold withering treatments. KEGG Annotation of DEPs Identified in the Heat and Cold Withering Treatments. To comprehend the canonical pathways, all DEPs were investigated through the KEGG. A total of 407 and 356 DEPs were annotated and allocated to 103 and 103 biological channels in heat and cold withering treatments, respectively. All annotated KEGG pathways were further filtered and enriched with P values below the 0.05 threshold as significant. In heat withering, a total of 20 KEGG pathways were identified and classified into two categories of “metabolism” and “genetic information processing” (Figure 4A). In cold withering, 17 KEGG pathways were identified that were commonly found in heat withering and also classified into two main categories of “metabolism” and “genetic information processing” (Figure 4B). The common four pathways belonging to “global and overview maps” subtypes contain the largest number of DEPs. One DEP identified in cold withering corresponds to caffeoylCoA O-methyltransferase (CCoAOMT, EC: 2.1.1.104). This enzyme is particularly important for lignin biosynthesis.39 One downregulated DEP corresponding to xanthine dehydrogenase (XDH, EC: 1.17.3.2) is related to caffeine metabolism in both

RESULTS Identification of DEPs in the Heat and Cold Withering Treatments of Postharvest Tea Leaves. The tender shoots containing first leaf, second leaf, third leaf, and immature stem of tea plant were picked and then subjected to 4 h withering treatments under 25, 38, and 4 °C, respectively; 32.88% (S1), 10.12% (CK), and 12.29% (S2) moisture of fresh weight were evaporated. In three samples of 25, 38, and 4 °C treatments, a total of 2486 proteins were acquired by iTRAQ technology. In heat withering, a total of 635 DEPs consisting of 445 upregulated proteins (S1: CK ratio > 1.5, q value < 0.01) and 190 downregulated proteins (S1: CK ratio < 0.67, q value < 0.01) were detected (Figure 1A). In cold withering (4 °C), a total of 566 DEPs consisting of 452 upregulated proteins (S2: CK ratio > 1.5, q value < 0.01) and 114 downregulated proteins (S2: CK ratio < 0.67, q value < 0.01) were detected (Figure 1B). After removing the duplicate, a total of 787 DEPs were obtained in heat and cold withering. A Venn diagram count of DEPs showed that 340 (43.2%) proteins either in heat or in cold withering treatments were upregulated and 65 (8.3%) proteins either in heat or in cold withering treatments were downregulated. Limited numbers of the proteins were upregulated in heat withering and downregulated in cold withering or upregulated in cold withering and downregulated in heat withering (Figure 2). On the basis of the fold change of expression values, the top and bottom 10 DEPs of upregulated and downregulated are listed in Table 2. The highest expressed protein was an ATPdependent protease La domain-containing protein in both heat and cold withering treatments. The protein is a member of the Lon protease family, which responds to the degradation of

Figure 2. Venn diagram of DEPs identified from tea leaves under withering treatments. 7549


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Table 2. List of the Top and Bottom 10 DEPs of Upregulated and Downregulated in Heat and Cold Withering Treatments DEPs (S1/CK) ID (in grape) top 10 DEPs

F6H1K6

A5C2R3

Q546P8 D7SJJ0 D7TDF1 D7SMK4 F6H9Y5 A5C5 V4

7.11 7.02 6.46 6.23 5.98 −2.41

D7TDF1 Q546P8 D7SJJ0 A5BIN1 D7T8L4 D7FBA7

beta carbonic anhydrase 2, chloroplastic basic endochitinase B nonspecific lipid-transfer protein 3 UDP-glucuronic acid decarboxylase 5 protein VAC14 homologue 40S ribosomal protein S9-2

6.91 6.74 6.44 6.26 6.22 −1.91

A5BE55

GDSL esterase/lipase APG

−2.59

D7TPX8

−2.07

D7UBN6 F6HP07

glycoside hydrolase family 2 protein serine/threonine-protein phosphatase PP1 isozyme 7 peroxisome biogenesis protein 22

−2.76 −2.94

A5AKV9 F6GUN5

dolichyl-diphosphooligosaccharide-protein glycosyltransferase aspartyl protease family protein 2 60S ribosomal protein L4-1

−3.39

F6HGJ5

−2.41

E3 ubiquitin-protein ligase UPL2 transducin/WD40 domain-containing protein translationally controlled tumor protein 1 casein kinase 1-like protein 6 coatomer subunit epsilon-1

−3.59 −4.20

D7T4J6 F6HP07

−4.56 −6.21 −6.52

F6GV40 F6HN96 D7U0U7

L10-interacting MYB domain-containing protein 40S ribosomal protein S4-3 serine/threonine-protein phosphatase PP1 isozyme 7 Hhstone H2B.11 60S ribosomal protein L23a-2 transducin/WD40 domain-containing protein

D7TEP0 F6H151 D7U0U7 D7U9U6 F6HZM0 D7TKQ8

F6H1K6

7.84

A5C9F4

7.68 7.55

D7SMK4 F6GYV0

log 2 (fold change)

7.51

F6GYV0

8.12

protein name

UDP-glucuronic acid decarboxylase 5 glycerol-3-phosphate dehydrogenase SDP6, mitochondrial eukaryotic translation initiation factor 2 (EIF-2) family protein basic endochitinase B nonspecific lipid-transfer protein 3 beta carbonic anhydrase 2, chloroplastic probable galacturonosyltransferase 9 cyclin-dependent kinase C-1 N-carbamoylputrescine amidase

A5BIN1 A5C9F4

ATP-dependent protease La domaincontaining protein 25.3 kDa heat shock protein, chloroplastic

ID (in grape)

ATP-dependent protease La domaincontaining protein glycerol-3-phosphate dehydrogenase SDP6, mitochondrial probable galacturonosyltransferase 9 eukaryotic translation initiation factor 2 (EIF-2) family protein orange protein

E0CVB4

bottom 10 DEPs

protein name

DEPs (S2/CK) log 2 (fold change)

8.42 7.72 7.61 7.40 7.24

−2.14 −2.19

−2.42 −2.43 −3.64 −4.00 −4.49

most catechins biosynthesis-related proteins except CsPAL and CsLAR. CsLAR did not interact with other proteins. Dynamic Changes of Catechins Contents in Postharvest Tea Leaves under Different Temperature Treatments. The “biosynthesis of secondary metabolites” pathway was specifically observed. The results showed that several DEPs participate in the catechins metabolic pathways. Three DEPs involved in flavonoid biosynthesis were identified in heat and cold withering treatments. The DEPs identified in heat withering correspond to three enzymes, i.e., CHI (EC: 5.5.1.6), F3H (EC: 1.14.11.9), and ANR (EC: 1.3.1.77). These enzymes were vital to catechins biosynthesis in tea plant. All of the three proteins were upregulated in high- and lowtemperature treatments (Figure 6 and Table 3). CHI and F3H were involved in the initial stage of the catechins pathway. ANR acted at the end of the catechins pathway. To further study the effects of different temperature treatments on the accumulation of catechins in postharvest tea leaves, the catechins contents were measured. The contents of monomeric catechins of tea leaves were detected by UPLC (Figure 7). The total amount of catechins was lowest under high-temperature treatment; however, the content of catechins was similar under low and room temperature treatments. In nongallated catechins (EC, GC, EGC), the catechins contents increased in the following order: high temperature < room temperature < low temperature. However, in C and galloylated catechins (ECG, CG, GCG, EGCG), the catechins contents decreased in the following order: room temperature > low temperature > high temperature. Gallated catechins (ECG, CG, GCG, EGCG) were higher than nongallated catechins (C,

withering treatments. Five and seven upregulated DEPs were related to terpenoid backbone biosynthesis in heat and cold withering treatments, respectively. Five DEPs identified in heat withering correspond to acetoacetyl-CoA thiolase (AACT, EC: 2.3.1.9), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR, EC: 1.1.1.267), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (MEPCT, EC: 2.7.7.60), isopentenyl diphosphate isomerase (IDI, EC: 5.3.3.2), and geranylgeranyl diphosphate reductase (CHLP, EC: 1.3.1.83). In addition to the above five enzymes, other two DEPs correspond to (E)-4hydroxy-3-methylbut-2-enyl-diphosphate synthase (GcpE, EC:1.17.7.1/1.17.7.3) that was only identified in cold withering. Except for “global and overview maps” subtypes, other KEGG pathways belong to “carbohydrate metabolism”, “energy metabolism”, “amino acid metabolism”, “metabolism of cofactors and vitamins”, “translation”, and “folding, sorting, and degradation” subtypes. Overall, the upregulated DEPs in common pathways were predominant relative to downregulated DEPs in either heat withering or cold withering treatment. Protein−Protein Interaction (PPI). The proteins in the catechins biosynthesis were analyzed by a string network based on the orthologs of Arabidopsis (Figure 5). Line colors indicated the types of interaction evidence. Most of these proteins can interact with each other. In particular, CsCHI can interact with seven enzymes that are involved in catechins biosynthesis, including CsPAL, CsC4H, CsCHS, CsANS, CsF3H, CsF3′5′H, and CsDFR. CsF3H interacted with eight enzymes, CsPAL, CsC4H, CsCHS, CsANS, CsF3H, CsF3′5′H, CsDFR, and CsCHI. CsANR could interact with 7550


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Figure 3. GO classification of DEPs identified from tea leaves under withering treatments. Top 20 terms of “biological process”, “cell component”, and “molecular function” were counted according to the P value in ascending sort order. (A) Heat withering (S1/CK). (B) Cold withering (S2/ CK).

those genes and their respective proteins. CsMPK gene expression in heat withering was upregulated, while CsMPK protein expression was downregulated. The expression levels of CsHSP1, CsDFAD, and CsMPK were upregulated in cold withering, while these genes corresponding proteins were downregulated. Although the expression trend of CsHSP1 was consistent with its protein in heat withering, the expression level of CsHSP1 was 755 times the control group. Expression Levels of Catechins Related-Genes under Different Temperature Treatments. In this study the expression levels of 17 catechins-related genes were detected by RT-qPCR (Figure 10). Most genes were upregulated at high- and low-temperature treatments. Under heat withering, the expression levels of CsPAL, Cs4CL, CsCHI-2, and CsF3′5′H were strongly suppressed, while under cold withering, only CsPAL and CsLAR-2 were suppressed. Except CsLAR-2, the expression levels of the other catechins-related genes were significantly higher in cold withering than that in

EC, GC, EGC) under the three different temperature treatments (Figure 8). Comparative Analysis of Protein and Gene Expression Profiles Response to Temperature. To validate the DEP expressions, the gene expression levels of 11 DEPs in response to temperature were detected by RT-qPCR (Figure 9 and Table 4). The results showed that expression levels of 9 genes were accordance with iTRAQ results in heat or cold withering treatments. For secondary metabolism, 8 genes were suppressed in heat withering and 8 genes were induced in cold withering. Expression levels in heat withering display obvious differences between gene and protein, while genes expression levels in cold withering were relatively consistent with protein expression. Moreover, we investigated four temperature-sensitive genes, which encode HSP1, HSP2, delta (8)-fatty-acid desaturase (DFAD), and mitogen-activated protein kinase (MPK). There was no good correlation between the expression trends of 7551


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Figure 4. KEGG classification of DEPs identified from tea leaves under withering treatments. P values below the 0.05 threshold. (A) Heat withering (S1/CK). (B) Cold withering (S2/CK).

Correlation Analysis of Catechins Contents and Expression Levels of Related Genes and DEPs. The correlation coefficient of catechins contents and expression levels of catechins-related genes and DEPs were calculated by

heat withering. CsANR-1, CsANR-2, and CsANR-3 belong to the same family and showed similar expression trends. However, CsLAR and CsLAR-2 belong to the same family and showed different expression trends. 7552


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Figure 5. Protein−protein interaction analysis among enzymes in the catechins metabolism pathway.

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DISCUSSION Proteomic analysis has been used in diversified plants. To date, research on postharvest tea leaves under different temperatures has been relatively lacking. Proteomic analysis can help us better understand the molecular mechanisms underlying postharvest tea leaves. Here, we discovered that some biological processes were involved in postharvest tea leaves in response to high- and low-temperature treatments (Figure 11). DEPs not only participated in catechins metabolism but also were involved in the biological basis of leaf growth and development as well as the role of endogenous hormones in leaf development and senescence. These results provided new thoughts on the effects of different temperature treatments and dynamic changes catechins content in postharvest tea leaves. DEPs Were Involved in the Response to Acute Temperatures in Postharvest Tea Leaves. Numerous DEPs were annotated to the temperature-related terms of GO biological process, such as “heat acclimation”, “response to heat”, “cold acclimation”, and “response to cold” terms. These proteins play vital roles in heat or cold stimulus. HSP is a type of stress response protein, which is indispensable for all organisms to survive under extreme stimulation.36 The formation of HSP is sufficient to be activated by heat and

Figure 6. Expression profiles of DEPs in the catechins pathway in tea leaves under different temperature treatments.

Pearson analysis (Tables 5 and 6). The correlations were analyzed from three aspects: the control, heat, and cold withering treatments. Most of the genes expression levels were positively correlated with the catechins content (except ECG +CG). The gene expression levels of CsCHI, CsF3H, and CsANR were positively correlated with the catechins content, while the protein levels of CsCHI, CsF3H, and CsANR were negatively correlated with the catechins contents, except the protein level of CsANR was positively correlated with the EGC.

Table 3. Expression Profiles of DEPs Involved in the Catechin Biosynthesis Pathway in Tea Leaves under High- and LowTemperature Treatments treatment high temperature

low temperature

accession no.

protein symbol

fold change

log 2 (fold change)

identity

Uniprot

chalcone isomerase

P51117

CsCHI

2.128138

1.089592

66.24

P41088

flavanone 3-hydroxylase anthocyanidin reductase chalcone isomerase

A5BJF6

CsF3H

2.208005

1.142743

70.96

Q9S818

Q7PCC4

CsANR

3.162277

1.660964

64.86

Q9SEV0

P51117

CsCHI

1.169499

0.225891

66.24

P41088

flavanone 3-hydroxylase anthocyanidin reductase

A5BJF6

CsF3H

1.419057

0.504933

70.96

Q9S818

Q7PCC4

CsANR

2.355049

1.235757

64.86

Q9SEV0

protein name

7553

Uniprot_URL http://www.uniprot.org/uniprot/ P41088 http://www.uniprot.org/uniprot/ Q9S818 http://www.uniprot.org/uniprot/ Q9SEV0 http://www.uniprot.org/uniprot/ P41088 http://www.uniprot.org/uniprot/ Q9S818 http://www.uniprot.org/uniprot/ Q9SEV0 DOI: 10.1021/acs.jafc.9b01705 J. Agric. Food Chem. 2019, 67, 7547−7560

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Figure 7. UPLC profiles of postharvest tea leaves under different temperature treatments. (A) Standard sample containing all eight catechins. (B) Under 38 °C temperature treatment. (C) Under 4 °C temperature treatment. (D) Under 25 °C temperature treatment. GC, gallocatechin; EGC, epigallocatechin; C, catechin; EC, epicatechin; EGCG, epigallocatechin gallate; GCG, gallocatechin gallate; CG, catechingallate; ECG, epicatechin gallate

Figure 8. Catechin contents in postharvest tea leaves under different temperature treatments. Values are the means of three independent experiments and are calculated as mg catechin equivalents per 1 g DW (mg/g). Error bars represent the standard deviation among three independent replicates. Data are mean ± SD of three independent replicates.

cold stresses.40 In this study we found that many upregulated DEPs belonging to HSPs were generated in heat and cold withering treatments, but more HSPs with higher expression were found in heat withering. In the KEGG pathway, several upregulated HSPs responding to heat and cold witherings were involved in “protein processing in endoplasmic reticulum”, indicating that HSPs may provide a protection for the cells through processing protein in endoplasmic reticulum. Except for HSPs, at least 90 DEPs were temperature-responsive

proteins. These proteins may provide full protection to cells of postharvest tea leaves in acute temperature stresses. DEPs Were Involved in Leaf Energy Metabolism. Energy metabolism is the biological basis of leaf growth and development.41 KEGG pathways showed that at least 40 DEPs were involved in “carbon fixation in photosynthetic organisms”, “photosynthesis”, and “photosynthesis-antenna proteins”. Most DEPs participated in energy metabolism turning out to be upregulated especially in “carbon fixation in 7554


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Figure 9. Comparative analysis of transcription and protein levels of DEPs. CsCCoAOMT: caffeoyl-CoA O-methyltransferase. CsXDH: xanthine dehydrogenase. CsDXR: 1-deoxy-D-xylulose-5-phosphate reductoisomerase. CsAACT: acetoacetyl-CoA thiolase. CsMEPCT: 2-C-methyl-Derythritol 4-phosphate cytidylyltransferase. CsIDI: isopentenyl diphosphate isomerase. CsGcpE: (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase. CsHSP: heat shock protein. CsDFAD: delta(8)-fatty-acid desaturase. CsMPK: mitogen-activated protein kinase. Error bars represent the standard error. Interval at Y axis is omitted from 7 to 754.

Table 4. Genes for RT-qPCR in C. sinensis Transcriptome gene name

full name

ID (in grape)

ID (inC. sinensis)

CsCCoAOMT CsXDH CsDXR CsAACT CsMEPCT CsIDI CsGcpE CsHSP1 CsHSP2 CsDFAD CsMPK6

caffeoyl-CoA-O-methyltransferase xanthine dehydrogenase 1-deoxy-D-xylulose 5-phosphate reductoisomerase acetoacetyl-CoA thiolase 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase isopentenyl diphosphate isomerase (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase heat shock protein heat shock protein delta(8)-fatty-acid desaturase mitogen-activated protein kinase

D7TPG4 F6GU50 D7SHS1 F6HHQ7 D7TGK2 F6GX19 D7SKY1 A5B868 F6HCT7 F6H3N5 F6HAX6

Tea_T1_Unigene_BMK.70625 Tea_T2_Unigene_BMK.50808 Tea_T4_Unigene_BMK.59750 Tea_T4_Unigene_BMK.64624 Tea_T1_Unigene_BMK.78133 Tea_T3_Unigene_BMK.31611 Tea_T1_Unigene_BMK.76928 Tea_T1_Unigene_BMK.22600 Tea_T1_Unigene_BMK.73227 Tea_T1_Unigene_BMK.66879 Tea_T1_Unigene_BMK.78880

photosynthetic organisms” and “photosynthesis-antenna proteins” pathways. In “photosynthesis”, several DEPs corresponding to the modules of photosystem II, cytochrome b6/f complex, and F-type ATPase were downregulated in heat withering relative to in cold withering. Those results indicated that the metabolites of tea leaves after harvesting were damaged in a higher level under high-temperature treatment than that low-temperature treatment. Endogenous Hormone-Related DEPs Were Activated or Inhibited in Acute Temperatures of Postharvest Tea Leaves. Endogenous hormones are widely involved in leaf development and senescence.42 In this study, DEPs participated in the biological processes of five endogenous hormones (i.e., auxin, abscisic acid, ethylene, cytokinin, and brassinosteroid) in GO annotation. Most of these DEPs were involved in the signaling of abscisic acid, ethylene, cytokinin, and brassinosteroid and were upregulated in heat and cold withering treatments. Abscisic acid plays an important role in leaf senescence and abscission, and cytokinin promotes cell differentiation and growth. From the perspective of the phenotype of postharvest tea leaves under high- or lowtemperature witherings, endogenous hormones may play roles

in maintaining the balance of cellular senescence. In auxin signaling, most DEPs were downregulated in heat withering, whereas most DEPs were upregulated in cold withering. It indicated that heat withering may suppress auxin signaling and accelerate cellular senescence of postharvest tea leaves. DEPs Were Involved in the Suppression of Cellular Development in Acute Temperatures of Postharvest Tea Leaves. GO terms revealed that the expressions of most DEPs were inhibited in at least nine mitosis-related biological processes, including “mitotic sister chromatid cohesion”, “mitotic nuclear division”, “mitotic sister chromatid segregation”, “mitotic cell cycle process”, “mitotic cell cycle”, “mitotic recombination”, “mitotic cytokinetic process”, “mitotic cytokinesis”, and “regulation of mitotic cell cycle”. It indicated that acute temperature stress could suppress cell’s mitosis of postharvest tea leaves. Plant cell metabolism is a process based on RNA transport and degradation.43,44 Most DEPs involved in RNA transport and degradation were upregulated in heat and cold witherings. We speculated that acute temperature stress may accelerate RNA metabolism to accelerate the withering metabolism of postharvest tea leaves. 7555


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Figure 10. Gene expression profiles of catechins biosynthesis genes in postharvest tea leaves under different temperature treatments. Each bar was calculated among three independent replicates. Data are the mean ± SD of three replicates. Different lowercase letters indicate significant differences at P < 0.05.

Table 5. Correlation Analysis between Catechins Content and Expression Levels of Catechins-Related Genesa CsPAL CsC4H Cs4CL CsCHS CsCHI CsCHI-2 CsF3′H CsF3H CsF3H-2 CsF3′5′H CsDFR CsANS CsANR CsANR-2 CsANR-3 CsLAR CsLAR-2

GC

EGC

C

EC

EGCG+GCG

ECG+CG

TC

−0.078 0.893 0.995 0.891 0.835 0.778 0.903 0.891 0.886 0.969 0.874 0.883 0.889 0.911 0.91 0.893 −0.998*

−0.37 0.986 0.92 0.985 0.961 0.557 0.99 0.987 0.985 0.849 0.98 0.984 0.986 0.993 0.993 0.987 −0.932

0.628 0.341 0.792 0.336 0.23 0.997* 0.362 0.348 0.339 0.88 0.315 0.331 0.344 0.391 0.388 0.353 −0.777

−0.002 0.857 1.000* 0.854 0.791 0.823 0.868 0.855 0.85 0.985 0.836 0.845 0.853 0.878 0.876 0.857 −1.000**

0.811 0.08 0.603 0.074 −0.036 0.941 0.103 0.08 0.07 0.717 0.045 0.061 0.075 0.126 0.122 0.084 −0.576

0.971 −0.299 0.26 −0.304 −0.407 0.746 −0.277 −0.302 −0.311 0.402 −0.335 −0.319 −0.306 −0.258 −0.261 −0.297 −0.225

0.607 0.366 0.808 0.361 0.256 0.999* 0.387 0.366 0.357 0.889 0.333 0.349 0.362 0.409 0.406 0.371 −0.789

Correlations were determined by Pearson correlation coefficient (r) analysis. * indicates significance at p < 0.05, and ** indicates significance at p < 0.01. a

Proteasome is a type of protein complex, which mainly has functions in protein degradation.45 Except for Rpn2 of the base module downregulated in heat withering, DEPs corresponding

to the base and 20S colre particle modules of proteasome were upregulated in heat and cold witherings. DEPs corresponding to Rpn5, Rpn6, and Rpn7 of the lid module were down7556


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Journal of Agricultural and Food Chemistry Table 6. Correlation Analysis between Catechins Content and Expression Levels of Catechins-Related DEPsa CsCHI CsF3H CsANR

GC

EGC

C

EC

EGCG+GCG

ECG+CG

TC

−0.648 −0.475 −0.174

−0.392 −0.193 0.126

−0.994 −0.95 −0.803

−0.703 −0.541 −0.248

−0.988 −0.999* −0.932

−0.857 −0.945 −1.000*

−0.991 −0.941 −0.786

Correlations were determined by Pearson correlation coefficient (r) analysis. * indicates significance at p < 0.05.

a

Figure 11. Simplified diagram of some biological processes under different temperature treatments.

faster than degradation above 44 °C.13 In higher plant, the levels of secondary metabolites may be involved in energy metabolism. Under the change of ambient temperature, plants will invoke more energy metabolism to participate in and maintain normal physiological metabolism of itself.49 DEPs Were Involved in the Catechins Metabolic Pathway in Acute Temperatures of Postharvest Tea Leaves. In this study, three DEPs (CHI, F3H, and ANR) involved in catechins biosynthesis were identified. CHI catalyzes chalcone to naringenin.9 F3H catalyzes flavanones to produce dihydroflavonols.10 ANR acts at the end of the catechins pathway.11 The expression profiles of all three DEPs involved in catechins biosynthesis were upregulated both in heat and in cold withering treatments, while the catechins contents declined under 38 °C treatment. Catechins readily oxidize at high temperatures. The catechin degradation rate may be higher than the increase of catechin metabolic enzyme activity in high temperatures of postharvest tea leaves.50 This may be the main reason for inconsistent catechins contents and proteins expression. The inconsistency findings were similar to the previous studies.51,52 The XDH (xanthine dehydrogenase) was also changed after withering treatments, which is related to caffeine metabolism. Caffeine is also an

regulated in heat withering, whereas DEPs corresponding to Rpn5 was downregulated and DEPs corresponding to Rpn6 was upregulated in cold withering. The lid module of proteasome plays a role in protein deubiquitination.46 The base module unfolds the proteins without ubiquitin chains and transports these proteins to the degradation module of the 20S core particle.47 We speculated that acute temperature stress promotes protein degradations of postharvest tea leaves; heat withering may affect protein deubiquitination. Dynamic Changes of Catechins Contents in Acute Temperatures of Postharvest Tea Leaves. Catechins were the main ingredient of tea polyphenols in tea leaves.48 Fresh tea leaves were picked and spread for several hours for withering, which was an important procedure for making black and oolong tea, etc. However, the withering of tea leaves was affected by the changing ambient temperature. In this study, the catechins content of postharvest tea leaves changed after acute temperature treatments. The catechins contents declined more in postharvest tea leaves under 38 °C treatment than that in 25 and 4 °C treatments. That may be due to the degradation of catechins in postharvest tea leaves during heat treatment, as catechins readily oxidize at high temperatures.13 In tea processing, the degradation of catechins was more profound below 44 °C, while the epimerization from GCG to EGCG was 7557


Journal of Agricultural and Food Chemistry important secondary metabolite of tea plant and contributes to the quality of tea.53 In this study we mainly focused on the effects of different temperatures on catechins of postharvest tea leaves. In the future we should also pay attention to caffeine metabolite of postharvest tea leaves under different temperature withering. The changes of proteins may not only be adaptive but also be their own physiological regulation. In previous studies, Han and colleagues found that some proteins related to lignin and protection were regulated by high temperature.49 With the increase of ambient temperature, plants will invoke more and more protection mechanisms to participate in and maintain the normal physiological metabolism of itself.49 Proteins relating to carbohydrate and energy metabolism induced by cold stress may contribute to the effective carbon flux and adequate energy supply in celery leaves. It revealed that plants may need to produce more energy to fight with cold stress.54 Gene Expression Profiles in Postharvest Tea Leaves under Different Temperature Treatments. The expression levels of 17 catechins-related genes were investigated. Most of the genes were upregulated at both low and high temperature. This is consistent with the upregulation of protein levels. The expression levels of most genes were higher in cold withering than that in heat withering. The protein expression levels were higher at high temperature than that at low temperature, which were not consistent with gene expressions. This is a complex process, which may account for post-transcriptional and post-translational regulation. Almost all genes (except for CsPAL and CsLAR-2) were positively correlated with the catechins content (except for ECG+CG) under temperature treatments witherings. CsLAR and CsLAR-2 belong to the same family and showed differential expression trends. Some studies revealed that LAR could regulate the formation of catechins monomers with different structures.50 We inferred that different LAR genes might perform distinct functions in the catechins biosynthesis pathway. The expression level of genes (CsCHI, CsF3H, and CsANR) has inconsistent trends with the proteins levels of DEPs. Protein expression was the comprehensive result of multistep regulation, which was a complex process and might account for post-transcriptional and post-translational regulation. Therefore, there is no linear correlation from the genetic to the phenotypic level.55

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ABBREVIATIONS USED

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REFERENCES

ANR, anthocyanin reductase; ANS, anthocyanidin synthase; C, catechin; C4H, cinnamate 4-hydroxylase; CG, catechingallate; CHI, chalcone isomerase; CID, collision-induced dissociation; DEPs, differentially expressed proteins; DFR, dihydroflavonol 4-reductase; EC, epicatechin; ECG, epicatechingallate; EGC, epigallocatechin; EGCG, epigallocatechin gallate; F3H, flavanone-3-hydroxylase; F3′H, flavonoid-3′-hydroxylase; F3′5’H, flavonoid-3′,5′-hydroxylase; FDR, false discovery rate; GC, gallocatechin; GCG, gallocatechin gallate; GO, gene ontology; HPLC, high-pressure liquid chromatography; IDA, information dependent acquisition; iTRAQ, isobaric tags for relative and absolute quantification; KEGG, Kyoto Encyclopedia of Genes and Genomes; LAR, leucoanthocyanidin reductase; LC-MS/MS, liquid chromatography electrospray ionization tandem mass spectrometry; PAL, phenylalanine ammonia lyase;; PPI, protein−protein interaction; PSPEP, proteomics system performance evaluation pipeline; RT-qPCR, quantitative real-time polymerase chain reaction; SCX, strong cation exchange; UPLC, ultrahigh-performance liquid chromatography

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b01705.

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Primers for RT-qPCR assay; primers of genes involved in the catechin biosynthesis pathway (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jing Zhuang: 0000-0001-6960-5217 Funding

The research was supported by the National Natural Science Foundation of China (31570691; 31870681). Notes

The authors declare no competing financial interest. 7558


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