Isobaric Tags for Relative and Absolute Quantitation Proteomic

Feb 21, 2017 - However, the response of proteomics in germinating barley to GA and abscisic acid (ABA) treatments is not thoroughly understood. In thi...
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iTRAQ-based quantitative proteomic analysis of germinating barley under GA and ABA treatments Yuqing Huang, Shengguan Cai, Jianbin Zeng, Dezhi Wu, and guoping Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04865 • Publication Date (Web): 21 Feb 2017 Downloaded from http://pubs.acs.org on February 22, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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iTRAQ-based quantitative proteomic analysis of germinating barley under

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GA and ABA treatments

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Yuqing Huang, Shengguan Cai, Jianbin Zeng, Dezhi Wu, Guoping Zhang*

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Department of Agronomy, Zhejiang Key Lab of Crop Germplasm, Zhejiang University,

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Hangzhou 310058, China

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*

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571 88982117).

Corresponding author (E-mail: [email protected], phone: +86 571 88982115, Fax: +86

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Key words: barley, gibberellin (GA), abscisic acid (ABA), germination, proteomics

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Abstract

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The degradation of starch in barley grains is a primary step of beer production. The addition

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of appropriate amount of GA promotes the production of fermentable sugars, beneficial to

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brewing industry. However, the response of proteomics in germinating barley to GA and

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ABA treatments is not thoroughly understood. In this study, iTRAQ (isobaric tags for relative

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and absolute quantitation) proteomics analysis was performed to illustrate the change of

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proteins in Tibetan wild barley XZ72 and XZ95 under GA and ABA treatments during

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germination. XZ72 had more proteins up-regulated than XZ95 under GA treatment, while

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under ABA treatments, XZ95 had more proteins up-regulated than XZ72. Concerning the

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proteins involved in energy metabolism under GA treatment, XZ72 had more proteins up-

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regulated than XZ95. Among the 174 proteins related to starch metabolism, 31 proteins

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related to starch hydrolysis, such as α-amylase, α-glucosidase and β-fructofuranosidase,

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showed higher relative abundance in control and GA treatment in XZ72 than in XZ95.

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Analysis of correlation between proteins and metabolites indicated that higher hydrolases

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activity is beneficial for the accumulation of fermentable sugars during germination. On the

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other hand, 26 starch synthesis-related proteins were up-regulated in XZ95 under ABA

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treatment. It may be suggested that GA-induced proteins act as accelerators of starch

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degradation while ABA-induced proteins inhibit starch degradation. The current results

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showed that XZ72 is highly capable of allocating the starch hydrolysing enzymes which play

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important roles in starch breakdown.

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1. INTRODUCTION

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Malting is the first step in brewing industry, and its process consists of optimized grain

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germination when hydrolysis enzymes are secreted from the aleurone layer and the starch is

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digested into fermentable sugars.1 The hydrolysis of starch to the fermentable carbohydrates

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is an important step of brewing, which is achieved by the concerted function of α-amylase, β-

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amylase, limit dextrinase and α-glucosidase.2 Starch is the main carbohydrate reserve in

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cereal seeds. The starch composition in barley (Hordeum vulgare L.) grains is around 70-75%

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amylopectin and 25-30% amylose.3

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Gibberellins (GA) and abscisic acid (ABA) are two most common hormones which

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function as regulators of plant development. Many studies on a wide variety of plants have

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shown that GA and ABA have a strong influence on seed dormancy and germination.4,5 The

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increase of ABA concentration leads to the accumulation of storage nutrients and seed

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dormancy.6 On the contrary, GA breaks seed dormancy and promotes germination.7 The

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balance of GA and ABA controls the shift between maturation and germination. During

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germination, GA is synthesized in the embryo and transferred to the aleurone layer, where it

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stimulates the production and development of hydrolytic enzymes. The enzymes are further

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transferred to the endosperm to catalyze the hydrolysis of starch.

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It is a common practice in the brewing process to add an appropriate dose of GA to

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germinating barley seeds so as to enhance both α-amylase and limit dextrinase (LD),

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thusaccelerating germination.8 The mechanism for induced expression of α-amylase genes by

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GA has been extensively studied. GA stimulates while ABA inhibits the synthesis of α-

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amylase by regulating the mRNA level. The transcription factor GAMYB, which regulates

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the expression of α-amylase gene was highly induced by the addition of GA. On the other

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hand, GA3-induced transcription of the α-amylase encoding genes was inhibited by ABA.9

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Although the functional and expressional analysis of the genes related to α–amylase has been

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well established, the changes of β-amylase, limit dextrinase and α-glucosidase during

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germination have not been thoroughly understood.

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Tibetan wild barley is a progenitor of cultivated barley and rich in genetic diversity.10 As

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shown in previous studies, wild barley displays higher variation in limit dextrinase inhibitor,

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β-amylase activity and phenolic acids.11-15 According to our previous results, the malting

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quality of XZ72 and XZ95 differed greatly.16 Application of GA increased sugars and amino

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acids concentrations of germinating barley grains, with more increase in XZ72 than in

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XZ95.16 More fermentable sugars result in higher yield of malt extract, which is beneficial for

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beer production. However, the underlying mechanism in the difference of these metabolites

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between XZ72 and XZ95 is still unclear.

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The ‘omic’ strategy, such as proteome and metabolome analysis, has been considered as

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powerful tools in analyzing complex biological concerns.17 Actually metabolome,

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transcriptome and proteome methods have been widely applied in the studies of seed

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germination.18,19 Jin et al.20 used two dimensional electrophoresis to analyze the protein

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difference between two malt barley genotypes with distinct filterability and identified 40

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proteins, most of which were hydrolases and pathogen-related proteins.20 The iTRAQ is a

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more powerful and sensitive approach for protein quantification on the basis of peptide

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labelling and quantification. With the capacity of up to 8 samples analyzed simultaneously,21

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it improves largely the throughput of 2D-gel based techniques.22 Although the iTRAQ-based

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proteomics has been widely applied in studies of abiotic stress23,24 and nutrient deficiency

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responses25, little research has been done on grain quality, including barley malt quality. In

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this study, we conducted iTRAQ analysis to identify the proteins responding to GA and ABA

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treatments during germination using two barley genotypes differing in malt quality.

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Journal of Agricultural and Food Chemistry

2. MATERIALS AND METHODS

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2.1. Malting procedure. Two Tibetan wild barley accessions, XZ72 and XZ95 were micro-

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malted using Joe White Malting System (Adelaide, SA, Australia). The malting procedures

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were as below: (1) Steeping stage: 5–8–8–12–4–5–2 h (wet–dry–wet–dry–wet–dry–wet); (2)

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Germination stage: 96 h at 16 °C. GA (0.5 mg/l) or ABA (0.5 mg/l) was added at the last step

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of wet stage during steeping. Seed samples were collected after steeping stage and 24 h, 48 h,

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72 h, 96 h after germination.

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2.2. Determination of enzyme activity and β-glucan content. The activities of α-amylase,

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β-amylase, β-glucanase and limit dextrinase, and β-glucan content were measured using

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Megazyme assay kits (Megazyme International, Bray, Ireland), according to the

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manufacture’s manual instruction.

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2.3. Protein extraction and digestion. Frozen barley grains after steeping and 96 h after

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germination were ground under liquid nitrogen and extracted in lysis buffer (8 M urea, 1%

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Triton-100, 65 mM dithiothreitol (DTT) and 0.1% Protease inhibitor Cocktail (Sigma)).26

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After centrifugation at 20000 g at 4 °C for 10 min, the supernatant was collected and

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precipitated with cold trichloroacetic acid (TCA) for 2h at -20 °C. The supernatant was

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discarded after centrifugation at 4 °C for 10 min. The final pellet was dissolved in 8 M urea,

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100 mM triethylammonium bicarbonate (TEAB, pH 8.0) buffer before treatment of 10 mM

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DTT to reduce the protein and 20 mM indole-3-acetic acid (IAA) at room temperature in

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darkness for alkylation. The protein solution was then diluted to less than 2 M urea with 100

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mM TEAB. Trypsin was added to a final trypsin/protein mass ratio of 1:50 and left overnight,

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followed by an additional digest at 1:100 ratio for 4 h. 27

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2.4. iTRAQ labelling. The above protein solution was attached to a Strata×C18 SPE

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column for desalination and were then vacuum-dried. The tryptic peptides were dissolved in

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0.5 M TEAB buffer and incubated with 8-plex iTRAQ labelling kit for 2 h at room

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temperature. One hundred micrograms proteins of XZ72 and XZ95 collected after steeping

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and germination were labelled with iTRAQ reporters.

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2.5. Peptide fractionation and quantitative proteomic analysis by LC-MS/MS. The

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labelled samples were fractionated by high pH reverse-phase HPLC system connected to an

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Agilent 300 Extend C18 column (5 µm particles, 4.6 mm internal diameter, 250 mm length).

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The peptides were dissolved in 0.1% formic acid and then loaded onto a reversed-phase pre-

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column (Acclaim PepMan 100, Thermo Scientific). A reversed-phase analytical column

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(Acclaim PepMan RSLC, Thermo Scientific) was used to perform peptide separation. The

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workflow for gradient separation was set as follows: from 6% to 22% solvent B (0.1% FA in

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98% ACN) over 24 min, 22% to 36% in 8 min and 80% in 4 min and then maintained at 80%

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for another 4 min at a constant flow rate of 280 nl/min on an EASY-nLC 1000 HPLC system.

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The peptides were analysed by Q ExactiveTM Plus hybrid quadrupole-Orbitrap mass

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spectrometer (Thermo Fisher Scientific).

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The resolution of Orbitrap detection was set to 70,000. The voltage for electrospray was set

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at 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the ion trap, and

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it was set to 500,000 ions. The mass spectra scan range was 350 to 1800. Dynamic exclusion

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duration in the MS survey scan was 30.0 s and 20 most intensive signals were fragmented by

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MS/MS.

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2.6. Data analysis. The raw MS/MS data was processed by Mascot search engine (version

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2.3.0, Matrix Science, London, U.K.). All searches were performed by Hordeum vulgare

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protein database (http://www.uniprot.org/taxonomy/?query=Hordeum+vulgare&sort=score)

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which contains 73,385 sequences. Carbamidomethyl, iTRAQ-8 plex (N-term) and iTRAQ-8

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plex (K) were set as fixed modification, and oxidation on Met was chosen as variable

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modifications. Mass error for precursor ions and fragment ions were set to 10 ppm and 0.02

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Da, respectively. Cleaving enzyme which was represented by trypsin/P was allowed up to 2

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missing cleavage. The filters for protein identification were: significance threshold of P