Systematic Identification and Analysis of Lysine Succinylation in

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Omics Technologies Applied to Agriculture and Food

Systematic identification and analysis of lysine succinylation in strawberry stigmata Xianping Fang, Ya Xin, Zheliang Sheng, Hui Liu, Aili Jiang, Fang Wang, Jian Yang, Xiaojun Xi, Qian Zha, Liqing Zhang, Liangying Dai, Chengqi Yan, and jianping chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02708 • Publication Date (Web): 27 Aug 2018 Downloaded from http://pubs.acs.org on August 28, 2018

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Journal of Agricultural and Food Chemistry

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Systematic identification and analysis of lysine succinylation in

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strawberry stigmata

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Xianping Fang1, 2, Ya Xin3, Zheliang Sheng4, Hui Liu3, Aili Jiang2, Fang Wang5, Jian Yang1,

4

Xiaojun Xi2, Qian Zha2, Liqing Zhang2, Liangying Dai6, Chengqi Yan5* and Jianping

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Chen1*

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1

Institute of Plant Virology, Ningbo University, Ningbo 315211, China.

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2

Institute of Forestry and Pomology, Shanghai Academy of Agricultural Sciences, Shanghai

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201403, China.

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3

Hangzhou Academy of Agricultural Sciences, Hangzhou 310024, China

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4

Institute of Mountain Hazards and Environment, Chinese Academy of Sciences, Chengdu

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610041, China. 5

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Institute of Biotechnology, Ningbo Academy of Agricultural Sciences, Ningbo 315040, China.

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College of Plant Protection, Hunan Agricultural University, Changsha 410128, China.

15 16

* Correspondence:

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Jianping Chen, 86-574- 87609779, [email protected];

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Chengqi Yan, 86-574-89184038, [email protected];

19 20 21 22 23 24 25 26 27 1

ACS Paragon Plus Environment


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Abstract: The various post-translational modifications of plant proteins have important

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regulatory roles in development. We therefore examined various modified proteins from

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strawberry stigmata and found that succinylation of lysine residues was the most abundant

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type of modification. We then subjected proteins from strawberry stigmata to an efficient

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enrichment method for succinylated peptides and identified 200 uniquely succinylated

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lysines in 116 proteins. A bioinformatics analysis revealed that these proteins are involved

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in important biological processes, including stress responses, vesicular transport, and

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energy metabolism. Proteomics, combined with immunoprecipitation and immunoblotting,

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revealed an obvious increase in succinylation of the assembly polypeptide 2 (AP2) and

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clathrin from 0.5 to 2 h after pollination, suggesting that succinylation is involved in the

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recognition of pollen-stigma signaling substances and vesicular transport. These results

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suggest that AP2/clathrin-mediated vesicular transport processes are regulated by lysine

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succinylation during pollen recognition.

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Keywords: strawberry stigmata, succinylation, proteomics, AP2, clathrin

42 43 44 45 46 47 48 49 50 51 52 2


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Introduction

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The successful completion of sexual reproduction in flowering plants relies on

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pollen-stigma interactions. The stigma of angiosperms screens pollens that falls onto it, and

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will usually recognize compatible pollen. Subsequently, the pollen absorbs water from the

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stigma and this hydration increases its metabolic activity. Although sometimes the pollen

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tube growth starts even in case of the incompatible or foreign pollen, the growth can be

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stopped later. Thus the screening process is the first step of recognition between pollen and

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pistil, and consists of a mutual exchange of signal substances between the two.1 The stigma

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plays the most important role in this process of exchange and recognition,2 which is mostly

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mediated by interactions between proteins.3 Some proteases in the stigma may be involved

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in processes of signal transduction, signal recognition and stress resistance,3 but their exact

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biological function is still not fully understood.4

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Flower stigmata can effectively promote pollen germination by allowing pollen tubes to

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enter their tissues, so the research work on stigmata is of great significance.5

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Comprehensive analysis on spatiotemporal expression profiles in stigmata on

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transcriptomic levels have been performed in Arabidopsis thaliana,6 Oryza sativa,7

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Nicotiana tabacum.8 Besides, Goldman et al. used a specific promoter of the secretory zone

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cells in tobacco stigmata to drive cytotoxin gene expression in the tobacco stigma. As a

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result, they obtained tobacco plants without secretory zone cells. This transgenic tobacco

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plant did not produce any secretion on the stigma surface, resulting in female sterility. 9

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Research by Kim et al. on the lily stigmata indicated that the chemocyanin protein in lily

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stigma extracts could guide the polarized growth of the pollen tube, and the existence of

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stigma cysteine-rich adhesin could enhance the guidance of chemocyanin to the polarized

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growth of the pollen tube.10

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Many cellular proteins need to be modified to some extent after expression to perform

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their respective functions.11 Protein post-translational modification (PTM) can play

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important roles in regulating protein activity, structure and function, and their significance

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is widely recognized.12-16 The study of PTM has become increasingly interesting but

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complex with the identification of many PTM types, including phosphorylation,

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ubiquitination, methylation, succinylation, sumoylation, etc.11, 17 Such studies are useful for

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unraveling functionality and in-depth studies of the regulatory processes of proteins with

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PTMs (as opposed to “naked” proteins) will become more and more critical.18 PTM study

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has the potential to reveal the diversity and active states of proteins as well as the mode and

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mechanisms of their functionality. Finally, these methods provide a basis for the study of

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the molecular function involved in plant development process.11 There are suggestions that

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several proteins are involved in the pollen-stigma recognition process, or assist pollen tubes

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to grow and stretch into stigma tissues.10 Studies on protein PTMs have important

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significance for revealing the biological function of the stigma and to identify the signal

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pathway(s) related to pollination recognition in the stigma. Some phosphorylated proteins

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have been identified in Oryza sativa pistil.19 Furthermore, research on Brassica napus

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stigmata also suggested that the armadillo repeat–containing 1 protein can ubiquitinate the

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exocyst complex Exo70A1, thus leading to the rejection of compatible pollen as the

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self-incompatibility response.20

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Succinylation is another important type of PTM15 and occurs frequently in bacteria (e.g.

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Escherichia coli and Mycobacterium tuberculosis),21,

22

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gondii)23 and mammalian cells (e.g. human and mouse).24 Succinylation of the bacterial

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polysaccharide can also play a vital role in nodule invasion and possibly nodule

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development.25 Studies of succinylation in plants began recently when 325 succinylation

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modification siteswere identified in Taxusmedia,26 and 347 succinylation modification sites

protozoans (e.g. Toxoplasma

4


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were identified in tomato plants by liquid chromatography-tandem mass spectrometry

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(LC-MS/MS).27 Some computational methods were also developed to identify the

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succinylated proteins and sites.28 Furthermore, it has been reported that the vesicular

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transport pathway has a positive effect on the directional pollen tube growth.29 The

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assembly polypeptide 2 (AP2) complex is a key player in clathrin-mediated vesicular

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transport process.30 Amazingly, we found that AP2 and clathrin are both highly

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succinylated after pollination in strawberry stigmata.

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To reveal which protein PTM is the most widespread in strawberry stigmata, we used

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high-quality modifiable pan antibodies (broad-spectrum antibodies against each PTM) with

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different protein modification types in western blotting (WB) studies to comprehensively

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screen for six common protein modification types: phosphorylation (tyrosine), acetylation,

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succinylation, ubiquitination, methylation and sumoylation. We then conducted functional

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analyses of the most highly expressed of these modified proteins, which have provided a

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model for the signal regulatory and molecular mechanisms underpinning the stigma-pollen

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interaction process.

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Materials and methods

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Strawberry stigmata collection and protein extraction

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The Fragaria ananassa cultivar Hongyu was obtained from the Hangzhou Academy of

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Agricultural Sciences, Zhejiang, China. Plants were cultivated as described.31 Briefly, plants

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were kept in a growth chamber under a 10-h light/14-h dark cycle at 30 °C under 150 mol

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m–2 s–1 light or at 26 °C in the dark with a relative humidity of 60%. On the day before

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flowering, we emasculated and bagged the flowers before dehiscence. For pollination, we

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tried to brush around to bring all pollen from the stamens into the pistils in the center of the

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flower. Fresh stigmata (300 in total) from 100 strawberry plants served as a biological 5



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replicate with three replicates used. Stigmata (2 g in total per replicate) were pulverized in

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liquid nitrogen using a mortar and pestle. The powder was then transferred to a 50-mL

128

centrifuge tube and solubilized in cold 10% (w/v) trichloroacetic acid/acetone containing 50

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mM dithiothreitol (DTT), 0.1% (w/v) Protease Inhibitor Cocktail Set VI (Merck Millipore,

130

Billerica, USA), and polyvinylpolypyrrolidone powder for 2 h at –20 °C. After

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centrifugation at 20,000  g at 4 °C for 10 min, the supernatant was discarded. The pellet

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was washed twice with cold acetone supplemented with 50 mM DTT and 1 mM

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phenylmethanesulfonyl fluoride (PMSF), then air dried and resuspended in 8 M urea

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containing 2 mM EDTA, 10 mM DTT and 0.1% (w/v) Protease Inhibitor Cocktail Set VI

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(Merck Millipore, Billerica, USA). The sample was sonicated three times on ice using a

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Scientz high-intensity sonicator. Debris was removed by centrifugation at 20,000  g at

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4 °C for 10 min. The supernatant was transferred into a new tube, and the protein content

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was determined using reagents from a 2-D Quant kit (GE Healthcare). Stigmata were

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collected at three time points (2 h, 0.5 h, and 0 h) before pollination and two time points (0.5

140

h and 2 h) after pollination. Only stigma protein at 0 h was used for the WB screening of the

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various PTMs and the immunoprecipitation (IP) and subsequent succinylome analysis.

142

Strawberry stigmata samples collected at all five different time points were used for IP and

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WB experiments of clathrin, AP2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)

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(Figure 1).

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Protein tryptic digestion

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Protein disulfides were chemically reduced with 10 mM DTT for 1 h at 56 °C and then

147

alkylated with 55 mM iodoacetamide for 45 min at room temperature in the dark. Next,

148

proteins were precipitated with three volumes of pre-chilled acetone for 30 min at –20 °C.

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After centrifugation at 13,000  g at 4 °C for 20 min, the pellet was dissolved in 0.5 M

150

triethylamonium bicarbonat (TEAB) and sonicated for 5 min. Following a second 6


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centrifugation step as described above, the supernatant was collected. Protein in the

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supernatant (~3 mg) was digested with trypsin (Promega) overnight at 37 °C (1:50

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trypsin-to-protein mass ratio).

154

Affinity enrichment of succinylated peptides

155

Affinity enrichment of succinylated peptides was performed as described.32 Briefly, to

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enrich for succinylated peptides, tryptic peptides, prepared as described above, were

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dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, pH 8.0, 0.5%

158

(w/v) NP-40) and incubated with a pre-washed pan-anti-succinylated lysines (SuKs)

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antibody conjugated to agarose beads (PTM-402, PTM Biolabs, Hangzhou, China) overnight

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at 4 °C with gentle shaking. The beads were carefully washed four times with NETN buffer

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and twice with distilled H2O. Bound peptides were eluted from the beads in 0.1% (v/v)

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trifluoroacetic acid. Eluted fractions were combined and vacuum-dried in a SpeedVac

163

(Thermo Fisher Scientific). The peptides were passed through C18 ZipTip resin (Millipore)

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and then subjected to LC-MS/MS.

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LC-MS/MS

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Peptides (1 μg) were dissolved in solvent A (0.1% (v/v) formic acid (FA) in ddH2O) and

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loaded directly onto a reversed-phase pre-column (Acclaim PepMap 100, C18, 3-m particle

168

size, 100 Å pore size, 75 m ×2 cm column diameter and length, Thermo Fisher Scientific).

169

Peptides were separated using a reversed-phase analytical chromatography column (Acclaim

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PepMap RSLC, C18, 3-m particle size, 100 Å pore size, 75 m × 15 cm column diameter

171

and length, Thermo Fisher Scientific) connected to an EASY-nLC 1000 UPLC system. The

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eluent system was a linear gradient of 5–20% solvent B (0.1% (v/v) FA in acetonitrile) over a

173

30-min period and then 20–35% solvent B for a 10-min period at a flow rate of 300 nL/min.

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The resulting peptides were subjected to MS/MS using a Q Exactive hybrid

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quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) as described below.

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The peptides were initially fragmented in a nanospray ionization source coupled to a Q

177

Exactive Hybrid Quadrupole-Orbitrap mass spectrometer for MS/MS. Parent peptide ions

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were detected in the Orbitrap at a resolution of 70,000 m/z. Peptides were selected for

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MS/MS using a 25% normalized collisional energy with a 12% stepped-normalized

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collisional energy, and the fragments produced from the low, medium, and high collisional

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energies were combined and detected simultaneously.33 Ion fragments were detected in the

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Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one

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mass spectral scan followed by 20 tandem mass spectral scans was applied for the 20 most

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intense precursor ions that had a threshold ion count of 3  104 in the MS survey scan and a

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15.0-s dynamic exclusion. The electrospray ionization voltage was 2.0 kV. An automatic

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gain control was used to prevent overfilling the ion trap; 105 ions were accumulated for

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generation of the MS/MS spectra. For the MS scans, the m/z range was 350 to 1600. For the

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MS/MS scans, the smallest m/z value included was 100.

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Database search

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The MS/MS data were searched using MaxQuant as part of the integrated Andromeda

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search engine (v.1.4.0.5). Tandem mass spectra were searched against the NCBI Fragaria

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vesca

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https://www.ncbi.nlm.nih.gov/protein/?term=txid101020%5BOrganism%5D) concatenated

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with a reverse-decoy database and sequences of common protein contaminants. The MS raw

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data files and corresponding search parameter files can be found on the ProteomeXchange

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Consortium34

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(http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD008475). Trypsin/P

198

was specified as the cleavage enzyme; at most, two missed cleavages, four modifications per

database

website

with

(23,319

the

dataset

sequences,

identifier

PXD008475

8


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peptide, and five charges were allowed. Mass error was set to 5 ppm for precursor ions and

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0.02 Da for the fragment ions. Carbamidomethylated cysteines were specified as a fixed

201

modification. Methionine oxidation and SuK residues were specified as variable

202

modifications. The false-discovery rate thresholds for proteins, peptides, and modification

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sites were taken as 0.01. The minimum peptide length was set to seven. We removed any of

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the SuKs that were identified with a localization probability (modification (K) probability)

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of < 0.75 and contaminant protein sequences.

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Protein annotation, classification and subcellular location prediction

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The Gene Ontology (GO) annotated proteome for the succinylated proteins was derived

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from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/). First, the IDs of the

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succinylated proteins were converted into the corresponding UniProt IDs and then mapped

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onto the GO IDs according to their protein IDs. When a succinylated protein could not be

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annotated according to a GO term in the UniProt-GOA database, InterProScan software was

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then used to attempt to annotate the GO function of the protein based on protein sequence

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alignment with proteins of known function.35 Then the succinylated proteins were classified

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by GO annotation according to the ‘biological process’ and ‘molecular function’ categories.

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The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to annotate the

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pathways associated with the succinylated proteins. First, the KEGG online service

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‘Automatic Annotation Server’ was used to annotate the KEGG database description of each

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protein.36 The annotation was then mapped onto the KEGG pathway database using the

219

KEGG online service tool KEGG mapper. WoLF PSORT was used for predicting subcellular

220

localization.37

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Functional enrichment analysis

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As noted above, GO function enrichment analyses of the three ontologies, namely

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‘biological process’, ‘cellular component’, and ‘molecular function’, and the KEGG pathway

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enrichment analyses were performed to gain further insight into the function and pathways of

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the

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bioinformation-analysis-tools

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(https://github.com/JonesMacke/Bioinformation-anlaysis-tools.git)

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operating systems and Perl language. All of protein annotation information from NCBI

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Fragaria vesca database was used as background for performing GO/KEGG enrichment

230

analysis. Fisher’s exact test was then used to check for enrichment or depletion (right-tailed

231

test) of specific annotation terms among the members of the resulting protein clusters.

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Derived p-values were further adjusted to address multiple hypothesis testing by the method

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proposed by Benjamini and Hochberg.38 Terms with adjusted p-value < 0.05 in each cluster

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were treated as significant.

succinylated

proteins.

GO/KEGG

analysis

were

performed

using

by

the

Unix-based

235

Analysis of sequences surrounding succinylated lysines

236

Motif-x software was used to characterize the amino acid sequences surrounding each SuK

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residue (10 residues upstream and downstream of the SuK were included) in all protein

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sequences to develop a consensus sequence for the SuK-containing sites. Protein sequences

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downloaded from the NCBI Fragaria vesca database were used as background database

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parameters in Motif-x, and all other Motif-x parameters were taken as their default settings.

241

Analysis of protein-protein interaction networks

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The protein-protein interaction (PPI) network containing the identified succinylated

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proteins was characterized using Cytoscape software (http://www.cytoscape.org/).39 The

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proteins involved in the interaction network were obtained from the STRING database

245

(https://string-db.org/).40 The species Arabidopsis thaliana of STRING database was used to 10


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perform PPI analysis by protein sequences alignment. STRING defines a metric called the

247

“confidence score” to define the confidence that an interaction is real. We included all

248

interactions with a confidence score ≥ 0.7 (high confidence) to develop the network, which

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was then was visualized using Cytoscape. A recently developed graphical, theoretical

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clustering algorithm, denoted "Molecular Complex Detection" (MCODE),41 can be used to

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identify densely connected regions in large PPI networks that may represent molecular

252

complexes, with MCODE being part of the plug-in tool kit for the network analysis and

253

visualization software Cytoscape. The nodal density is an important parameter that can be

254

used to evaluate the importance of a node in a particular network, and we therefore

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calculated this parameter for each SuK-containing protein.

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Immunoprecipitation, western blotting screening and validation

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WB screening analysis was performed to reveal which protein PTM is the most

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widespread in strawberry stigmata. The levels of AP2 and clathrin lysine succinylation were

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also detected by combination of IP with WB at three time points (2 h, 0.5 h, and 0 h) before

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pollination and two time points (0.5 h and 2 h) after pollination.

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Antibodies used are listed as follows: anti-phosphotyrosine antibody (PTM-702, PTM

262

Biolabs, Hangzhou, China), anti-acetyllysine antibody (PTM-105, PTM Biolabs, Hangzhou,

263

China), anti-succinyllysine antibody (PTM-401, PTM Biolabs, Hangzhou, China),

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anti-diglycine lysine antibody (PTM-1101, PTM Biolabs, Hangzhou, China), anti-mono-,

265

dimethyllysine antibody (PTM-602, PTM Biolabs, Hangzhou, China), anti-SUMO-1

266

(ab5316, Abcam, Cambridge, England) and anti-SUMO-3 (ab5317, Abcam, Cambridge,

267

England). Antibodies for detecting AP2, clathrin and GAPDH (reference) were raised in

268

rabbits

269

126-CKRGKRGLMEDRYSA-140,

270

Biotechnology, Hangzhou, China). Briefly, 0.5 mg of peptide fragments were emulsified

using

the

respective

peptide and

fragments

72-CMQTENLELKKLVYL-86,

18-CHRSQASCVGLQHSS-31

(HuaAn

11



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with an equal volume of Freund's complete adjuvant (#F5881, Sigma-Aldrich) with a

272

sonicator and administered subcutaneously to the back of the healthy New Zealand white

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rabbits at 4 sites (200 μL/site). Prior to the first immunization, about 5 mL of blood was

274

collected from the ear vein of each rabbit to obtain pre-immune serum, which was used for

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corresponding control experiments. Three booster injections were administered 3, 5 and 7

276

weeks later using 0.25 mg of peptide fragments but emulsified with an equal volume of

277

Freund's incomplete adjuvant (#F5506, Sigma-Aldrich). Seven days after the final booster

278

injection, the blood was collected from the marginal vein in the ear and the serum was

279

separated for subsequent experiments. The pre-immune and post-immune serums were

280

assayed via enzyme-linked immunosorbent assays (ELISA), and the high titer of serum were

281

then purified. The immunoreactivity and immunogenicity of purified serum was further

282

validated by ELISA and WB. The entire process of antibody development mainly includes

283

steps of immunogen preparation, immunization, serum collection, ELISA screening,

284

purification, further evaluation and validation (Supplementary Table S1).

285

IP analysis was performed using an IP kit (#11719394001, Roche) following the

286

manufacturer’s instructions. Briefly, 400 µg of protein from total cell lysate was incubated

287

with 2 µg of antibody and subsequent to this incubation 20 µg of protein-G agarose was

288

added. The immune complex was incubated at 4 °C for 1 h,

289

centrifugation at 12,500 g for 1 min and washed four times with the buffer (20 mM Hepes /

290

NaOH (pH 7.4), 150 mM NaCl, 1 mM Na3VO4, 10 mM EDTA, 0.02 % NP-40, 10 µg/mL

291

aprotinin10 µg/mL leupeptin and 1 mM PMSF). Protein solutions were subjected by

292

SDS-PAGE, and then probed in a PVDF membrane using AP2, clathrin, GAPDH or pan

293

PTMs antibodies in a 1: 1,000 ~ 1,500 dilution. The HRP-labelled goat anti-rabbit secondary

294

antibody was diluted to 1:2500, andthe bands were probed by an ECL kit (Multisciences

295

Biotech). The immunoblot bands were quantified by band density using an ImageQuant LAS

then recovered by

12


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4000 with ImageQuant TL 7.0 software (GE Healthcare Life Sciences, Piscataway, NJ).

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Each experiment was repeated two (soly WB screening) or three times (IP/WB).

298

Results

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Identification of lysine succinylation in strawberry stigmata

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Although WB analysis using various PTM-specific pan antibodies showed that

301

succinylation is the most evident type of modification among various PTMs

302

(phosphorylation (Tyr), acetylation, succinylation, ubiquitination, methylation and

303

sumoylation) in strawberry stigmata (Figure 2A), phosphorylation (Ser/Thr) level is not

304

determined in this study and it will be studied in our further research. Thus we cannot

305

ignore the possibility that phosphorylation (Ser/Thr) might be more evident than

306

succinylation. However, high abundance of succinylated level in strawberry stigma proteins

307

is an interesting phenomenon. Two hundred succinylation sites in 116 proteins were

308

successfully identified with high confidence (Figure 2B, Supplementary Table S2). The MS

309

proteomics dataset has been deposited into the ProteomeXchange Consortium34 via the

310

iProX partner repository under the dataset identifier PXD008475/IPX0001091000

311

(http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD008475). The MS

312

data were validated by determining the mass error of all identified peptides. The

313

distribution of mass errors was near zero, and most values deviated < 0.02 Da, indicating

314

that the mass accuracy of the MS data met the requirement of proteomics analysis (Figure

315

2C). The identified succinylated proteins involved in many aspects of plant tissues and

316

organs (Figure 2D),26, 27, 42, 43 suggesting that succinylation might have important regulatory

317

roles in different plant species.

318

Comparison between lysine succinylation and acetylation in strawberry plants

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Of various PTMs, acetylation has been extensively mapped in diverse organisms. We

320

compared the identified succinylation sites in strawberry stigmata of this study to our

321

previously reported acetylation sites in strawberry leaves.44 Interestingly, we found that a

322

number of 53 strawberry stigmata proteins (Figure 3A) harboring 66 succinylated sites

323

(Figure 3B) could be acetylated at the same site(s) in strawberry leaves. Two hundred

324

succinylated sites were mapped on 200 peptides and 116 proteins (Figure 3C). Among the

325

116 succinylated proteins, 83 (72%) had one succinylation site and 13 (11%) had two

326

succinylation sites (Figure 3D). All succinylated peptides had lengths of 7–26 amino acids

327

(Figure 3E). Similarly, 473 (69%) AcK proteins had one acetylated site (Figure 3D). The

328

identified acetylated peptides ranged from 7 to 29 amino acids in length (Figure 3E).

329

Functional characterization of lysine succinylated proteins

330

The transport related terms including vesicular transport, hydrogen transport and anion

331

transport, were obviously enriched (Figure 4A). In the cellular component category, the

332

cytoplasm and protein complex were obviously enriched (Figure 4A). KEGG pathway

333

enrichment analysis identified many enriched pathways related to energy metabolism,

334

including carbon metabolism, pyruvate metabolism, glycolysis/gluconeogenesis and the

335

citrate cycle (Supplementary Table S3).

336

Through GO function classification analysis, we found that biological process category,

337

stress response and transport related proteins were the major succinylated proteins

338

accounting respectively for 21%, 19%, 15% and 12% of all the lysine succinylated proteins

339

(Supplementary Figure S1A). In the molecular function analysis, proteins associated with

340

catalytic activity, binding, oxidoreductase and transporter activity accounted for nearly 60%

341

(21%, 15%, 13% and 10%, respectively) of all the identified succinylated proteins

342

(Supplementary Figure S1B). In the cellular component category, the proportions of

343

membrane, organelle and macromolecular complex related proteins were 34%, 31% and 14


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24%, respectively (Supplementary Figure S1C). The subcellular localization analysis of the

345

identified proteins suggested that most were predicted to be localized in the cytosol (43%)

346

(Supplementary Figure S1D), suggesting important roles for lysine succinylation in cytosol.

347

Moreover, many proteins were localized in the nuclei (20%).

348

We then compared the function category of the identified proteins in this study to the

349

previously reported succinylated proteins in other plants.26,

27, 42, 43

350

strawberry stigma succinylated proteins have a much higher percentage of transport process

351

(Figure 4B), oxidoreductase activity, transporter activity (Figure 4C), nuclear, plasma and

352

extracellular (Figure 4D) than the succinylated proteins of other four plants, indicating that

353

succinylation might be closely involved in the transport and nuclear signal transduction

354

process in strawberry stigmata.

We found that the

355

Sequence-motif analysis for the succinylated peptides

356

To identify possible speciﬁc sequence motifs proximal to the SuK residues, we examined

357

the relative frequencies of amino acids in specific positions of peptides containing 21

358

residues and centered around each SuK residue and then compared those positions with

359

those of 21-mers that did not contain an SuK. We identified four motifs that were

360

significantly enriched in SuK residues from 200 unique SuK-containing sites, which

361

accounted for 95% of the identified sites. The four consensus sequence motifs were FSuK,

362

ISuK, LSuK, and VSuK (Table 1 and Supplementary Figure S2).

363

Analysis of interaction networks of succinylated proteins in strawberry stigmata

364

To investigate how the succinylated proteins are related and how they are involved in

365

different interacting pathways, we constructed a PPI network for all identified succinylated

366

proteins using the STRING database and Cytoscape software (Figure 5). Using the

367

MCODE plug-in toolkit, we extracted several highly enriched interaction clusters from the 15



Page 16 of 36

368

complete interaction network. Most of the proteins were clustered into one of five groups,

369

including the TCA cycle (succinyl-CoA ligase, malate dehydrogenase, etc.), oxidative

370

phosphorylation (ATP synthase CF1 alpha subunit, ATP synthase subunit beta, etc.) and

371

glycolysis (dihydrolipoyl dehydrogenase 1, fructose-bisphosphate aldolase, etc.) (Figure 5).

372

Detailed information concerning the proteins involved in these networks is presented in

373

Supplementary Table S4.

374

Pollination leads to increased hypersuccinylation of AP2 and clathrin

375

Among the different transport processes, the vesicular transport process has the highest

376

degree of enrichment (Figure 4A). AP2 and clathrin are two major types of vesicular

377

transport related proteins and the four peptides from these two proteins are obviously

378

succinylated in strawberry stigmata (Supplementary Figure S3), so we selected these two

379

proteins for subsequent validation experiments. To examine the levels of AP2 and clathrin

380

succinylation in vivo, we immunoprecipitated endogenous AP2 and clathrin by IP from

381

strawberry stigmata at five time points before and after pollination and examined protein

382

succinylation by WB (Figure 6). Quantitation of three independent experiments confirmed

383

that succinylation of AP2 and clathrin is significantly higher in pollinated stigmata when

384

compared with non-pollinated stigmata (Figure 6). The succinylation level of AP2

385

markedly increased (2.3-fold change at 0.5 h) when the stigmata were pollinated manually.

386

In contrast to AP2, the level of clathrin succinylation slightly increased at 0.5 h, yet with a

387

subsequent significant increase at 2 h after pollination. However, these two proteins levels

388

didn’t exhibit an obvious change after pollination, indicating that hypersuccinylation of

389

AP2 and clathrin is closely associated with strawberry stigma-pollen recognition process.

390

Discussion

16


Page 17 of 36


391

Protein succinylation modification refers to the process of covalent binding of a

392

succinyl-group to lysine residues in an enzymatic or non-enzymatic manner,45 so protein

393

succinylation can lead to a substantial change in the chemical properties of many proteins.

394

Importantly, the succinylation at the lysine residue induces charge mutations that, in

395

physiological pH, range from +1 to -1, thereby promoting the structural and functional

396

adjustment of the matrix proteins.15 At present, many studies have confirmed that protein

397

modification is ubiquitous in prokaryotes and eukaryotes, suggesting that lysine

398

succinylation regulates many important cellular metabolic processes in organisms.23, 32, 46

399

In this present study, we found highly significant succinylation amongst the modified

400

strawberry stigmata proteins and identified 200 succinylation modification sites in 116

401

proteins. These abundant succinylated proteins work as stress tolerance response proteins,

402

vesicular transport proteins, energy and metabolism related proteins, or are related to

403

synthesis and modification (Figure 4A). Upon flowering of the strawberry plant, the stigma

404

is directly exposed to the outside, and the pollen tube can enter the stigma tissue. Many

405

succinylated proteins (peroxidase, ascorbate peroxidase, catalase isozyme 1, etc.) are

406

related to adverse resistance and stress response process.31 This suggests that succinylation

407

is likely an important part of the signal pathway of the self-defense response that would

408

enhance resistance of the stigma to pathogen invasion, and actively create a good

409

pollination environment. After pollen germination, the stigma tissue exchanges signals with

410

the pollen tube, and may provide the raw materials needed by cells for the elongation of

411

pollen tubes. It appears that the abundant expression of succinylation-modified proteins

412

related to energy and metabolism in stigmata ensures a smooth process.

413

Besides, our study also showed that lysine succinylation is involved in the Calvin cycle,

414

oxidative phosphorylation and glycolysis process. Similarly, many proteins associated with

415

these metabolic pathways have also been found succinylated in other plants (succinyl-CoA 17



Page 18 of 36

416

ligase, malate dehydrogenase, dihydrolipoyl dehydrogenase, etc.).26, 27, 42, 43 These findings

417

suggest that lysine succinylation has a positive effect on photosynthesis and the Calvin

418

cycle. Our PTM proteome data supports the conclusion that mature stigmata of strawberry

419

plants possess a vigorous metabolism. The stigma cells provide material needed for future

420

pollen-stigma recognition, pollen germination and pollen tube elongation via genetic

421

transcription as well as protein synthesis and modification.47

422

More importantly, compared with the distribution of the succinylated proteins in Taxus

423

media,26 Solanum lycopersicum,27 Oryza sativa42 and Brachypodium distachyon,43 the

424

higher distribution of biological process, molecular function and subcellular location in

425

strawberry stigmata is transport process, transport activity and nuclear/extracellular,

426

respectively (Figure 4B-D). Thus the succinylated proteins involving in transport response

427

and locating in nuclear or extracellular might have important biological functions in the

428

stigma. We identified 14 succinylated proteins involving in different transport processes,

429

and 10 of these proteins have transport activity. These transporter related proteins include

430

F-type H+-transporting ATPase, coatomer subunit beta-1-like, coatomer subunit beta-2-like,

431

clathrin, AP2 complex, ran-binding protein 1, ATP synthase, small ubiquitin-related

432

modifier 2-like, metal-nicotianamine transporter, etc. (Supplementary Table S2). The

433

interaction of pollens and stigmata requires the transport and transmission of signaling

434

agents. The fact that a large number of transporter related proteins are succinylated

435

indicates that succinylation might regulate the pollen-stigma recognition process.

436

Among the four highly enriched transport processes (vesicular transport, hydrogen

437

transport, regulation of anion transport and ion transport), the vesicular transport has the

438

highest enrichment degree (Figure 4A). Vesicular transport is not only a basic process of

439

life, but also an extremely complicated dynamic biological process, related to various

440

proteins and regulatory molecules, and employing processes that are highly conserved in 18


Page 19 of 36


441

higher eukaryotes. In addition, vesicular transport pathways are important for directional

442

pollen tube growth process.29 Studies on self-incompatible plants also indicated that

443

endocytosis,

444

pollination-recognition process.20,

445

clathrin-mediated vesicular transport process.30

446

exocytosis

and

vesicular 48-50

transport

are

all

important

for

the

The AP2 complex is a key player in

Thus, based on the previous reports, our proteomics study and the up-regulated

447

succinylation

448

AP2/clathrin-mediated vesicular transport process plays a vital role in the pollination

449

recognition process of strawberries. Molecules promoting the germination and polarized

450

growth of compatible pollen as well as those that suppress the germination and growth of

451

incompatible pollen are transferred to the cell surface via exocytosis. Simultaneously,

452

signal recognition molecules from the pollen surface are introduced into the endomembrane

453

system via endocytosis, completing the recognition process in the stigma outer layers cells.

454

We identified numerous proteins that are involved in the vesicular transport in strawberry

455

stigmata, including two kinds of the most essential vesicular coat proteins: coatomer and

456

clathrin (Figure 7 and Supplementary Table S2). Vesicular transport mediated by coatomer

457

is considered to mediate non-selective material transport. This includes transport from

458

endoplasmic reticulum to the Golgi apparatus, between Golgi vesicles, and from the Golgi

459

apparatus to plasma membranes. However, clathrin can not only mediate transport from the

460

Golgi apparatus to the lysosome, plant vacuole or to the outside of the plasma membrane,

461

but also transport foreign substances into the cytoplasm, or from the endosome to the

462

lysosome. The vesicular coat of clathrin is the grid structure constructed by clathrin fibers,

463

and many different adapter proteins fill the frame between the clathrin structure and

464

envelope. These proteins mediate the connection between clathrin and the envelope

465

transmembrane protein receptor (the transport carrier), and form the clathrin

level

of

AP2/clathrin

after

pollination,

we

speculate

that

19



Page 20 of 36

466

vesicle-integrated structure together with dynamin. It is well known that four adapter

467

proteins, AP1, AP2, AP3 and AP4, selectively combine with different receptor-transporter

468

complexes, enveloping the foreign substances and forming specific transport vesicles.

469

In our study, we identified high-level succinylation in clathrin and in the adapter protein

470

AP2 (Figure 6 and Supplementary Table S2). We speculate that the succinylation of

471

clathrin/AP2 may assist in driving the cytoplasmic membrane to form a capsule and capture

472

various transmembrane specific receptor proteins (these enable the vesicles to envelop

473

specific molecules) in the vesicular transport system. A combination of plasma membrane

474

receptor and specific ligands (such as pollen proteins or other informational molecules

475

secreted from the pollen cells), enables the stigma outer layers cells memberanes to be

476

selective in AP2/clathrin via succinylation. AP2/clathrin in un-succinylated state will not

477

combine with the receptor, and succinylated AP2/clathrin can be further combined with the

478

receptor-ligand complex to form the vesicles. Then the vesicles can be budded, released and

479

fused into the organelles membranes. Thus the signal substances in pollen cells are

480

successfully transported into the stigma cell to further complete the pollination recognition

481

process (Figure 7). So, through a mechanism of interaction and recognition between pollen,

482

informational molecules and strawberry stigma, the stigma is able to respond smoothly to

483

the up-stream signal and promote successful pollen germination and pollen tube elongation.

484

Succinylation as well as the process of directional vesicular transport likely start this

485

process.

486

In this work, by combining comprehensive IP/WB analysis with high-affinity enrichment

487

of succinylated peptides as well as very sensitive mass spectrometry and advanced

488

bioinformatic tools, we systematically defined the lysine succinylome in strawberry

489

stigmata. The identification of 200 SuK sites in 116 proteins expands the lysine

490

succinylome catalog, especially for non-model plants. Functional characterization of 20


Page 21 of 36


491

succinylated proteins indicated that lysine succinylation is involved in diverse biological

492

processes and are present in various cellular components having diverse functions. Clathrin

493

and AP2 are highly succinylated, suggesting that succinylation of vesicular transport

494

proteins is important for the stigma-pollen recognition process in strawberry. In addition,

495

motif analysis extracted four consensus sequence motifs. Our research reveals that

496

succinylation modification is likely to play an important regulatory role in the

497

pollen-stigma recognition process and thus provides an important direction for the study of

498

strawberry reproductive development. The major vesicular transporters AP2 and clathrin

499

are highly succinylated after pollination and are likely to regulate the transport of signaling

500

substances via succinylation modification. It would be more meaningful if we carry out the

501

quantitative succinylome analysis at all the time points, though we have made a

502

global succinylome analyses at 0 h and found out the possible candidate succinylation sites.

503

Our future research will perform succinyl-null or succinyl-mimic mutation (i.e., a

504

lysine-to-arginine change) of the corresponding sites by PCR reaction to observe the

505

functional phenotype of plants, and further determine the role of succinylation in the

506

pollen-stigma signal transduction process.

507

Abbreviations

508

PTM, post-translational modification; LC-MS/MS, liquid chromatography-tandem mass

509

spectrometry; WB, western blotting; AP2, assembly polypeptide 2; PMSF,

510

phenylmethanesulfonyl fluoride; IP, immunoprecipitation; GAPDH, glyceraldehyde

511

3-phosphate dehydrogenase; NCE, normalized collisional energy; GO, gene ontology;

512

KEGG, kyoto encyclopedia of genes and genomes; PPI, protein-protein interaction; ELISA,

513

enzyme-linked immune-sorbent assays.

514

Funding sources 21



Page 22 of 36

515

This research was supported by grants from the Hi-Tech Program (‘863’ Program) of

516

China, Ministry of Science and Technology (Grant 2014A0A603-15); the Scientific and

517

Technological Project of Hangzhou (20170432B14); the Key Program of Zhejiang

518

Provincial Foundation for Natural Science (LZ14C140001, LZ16C130002, NB2016C11017);

519

and the National Key Research and Development Program of China (2016YFD0200804).

520

Conflict of interest statement

521

522

The authors declare no conflict of interest. Acknowledgments

523

We thank M. J. Adams, Minehead, UK for his help in correcting the English of the

524

manuscript.

525

Supporting information

526

The supplementary materials including four supplementary tables and three

527

supplementary figures for this article can be found online at https://pubs.acs.org/. The mass

528

spectrometry proteomics dataset has been deposited to the ProteomeXchange Consortium

529

via the iProX partner repository under the dataset identifier PXD008475/IPX0001091000

530

(http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD008475).

531 532

References

533 534 535 536 537 538 539 540 541

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636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 25



Page 26 of 36

659

Figure captions

660

Figure 1. The work flow of screening, enrichment and identification of strawberry

661

stigmata lysine succinylome by LC-MS/MS. An integrated strategy for global mapping of

662

lysine succinylation in strawberry stigmata.

663

Figure 2. Western blotting screening and identification of strawberry stigmata lysine

664

succinylome. (A) Equal amounts of total protein lysates (20 μg) were extracted from the

665

strawberry stigmata. Coomassie blue staining was used for the loading control. WB

666

experiments using the pan anti-phosphotyrosine, anti-acetyllysine, anti-succinyllysine,

667

anti-diglycine lysine, anti-mono-, dimethyllysine, anti-SUMO-1 or anti-SUMO-3 antibodies

668

were performed to assess phosphorylation (PTyr), acetylation (AcK), succinylation (SuK),

669

ubiquitination (GG), methylation (Me) and sumoylation (SUMO-1 / SUMO-3) levels in the

670

stigma protein, respectively. Equal amounts of protein were loaded into each lane. Each

671

experiment was performed in duplicate. (B) Venn diagram analysis of all the lysine

672

succinylated peptides and proteins from three biological replicates. (C) The distribution of

673

succinylated peptides based on their mass error. (D) The number of succinylated proteins

674

and sites identified in this study compared with previous studies.

675

Figure 3. Profile of identified succinylated and acetylated sites, peptides and proteins in

676

strawberry plants. (A) Venn diagrams illustrating the overlap between the SuK and AcK

677

proteins. (B) Venn diagrams illustrating the overlap between the SuK and AcK sites. (C)

678

Numbers of SuK and AcK sites, SuK and AcK peptides and SuK and AcK proteins. (D)

679

Distribution of SuK and AcK peptides in one protein. (E) Distribution of SuK and AcK

680

peptides based on their length.

681

Figure 4. Characterization of strawberry stigmata lysine succinylome. (A) Enrichment

682

analysis of all the identified lysine succinylated proteins based on the classification of GO 26


Page 27 of 36


683

annotation in terms of biological process, molecular function and cellular component. The

684

distribution and function of succinylated proteins identified in strawberry stigmata compared

685

with previous reported plant tissues and organs based on biological processes (B), molecular

686

functions (C) and subcellular locations (D).

687

Figure 5. PPI network of all the lysine succinylated proteins. Different geometric shapes

688

indicate various significantly enriched sub-clusters. Most proteins were clustered into one of

689

five groups, including the TCA cycle, oxidative phosphorylationand glycolysis.

690

Figure 6. Pollination Leads to increased hypersuccinylation of AP2 and clathrin. WB

691

showing succinylation of AP2 and clathrin immunoprecipitated from strawberry stigmata

692

protein at three time points (2 h, 0.5 h, and 0 h) before pollination and two time points (0.5 h

693

and 2 h) after pollination. Integrated density values werecalculated and are shown relative to

694

strawberry stigmata at 0 h. Each experiment was repeated three times IP:

695

Immunoprecipitation; WB: Western blotting; AP2: Assembly polypeptide 2; GAPDH:

696

Glyceraldehyde 3-phosphate dehydrogenase; SuK: Succinylated lysine.

697

Figure

698

clathrin/AP2–mediated vesicular transport of signal molecules. AP2/clathrin in

699

un-succinylated state will not combine with the receptor, and succinylated AP2/clathrin can

700

be further combined with the receptor-ligand complex to form the vesicles. Then the vesicles

701

can be budded, released and fused into the organelles membranes of strawberry stigma outer

702

layers cells. Thus the signal substances in pollen cells are successfully transported into the

703

stigma cell to further complete the pollination recognition process.

7.

A

proposed

model

depicting

how

succinylation

promotes

the

704 705

27



Page 28 of 36

Table 1. Motif analysis of the identified succinylated peptides in strawberry stigmata. Motif

a

Foreground

Motif a

Score

Background

Matches

Size

Matches

Size

Fold Increaseb

.........FK..........

4.7

21

232

111

3495

2.85

.........IK..........

3.71

26

211

193

3384

2.16

.........LK..........

3.58

35

185

326

3191

1.85

.........VK.........

2.87

26

150

265

2865

1.87

Motif score, the probalility of the succinylated sites in some motifs and the value was set as -Log10(P

value). The confident cut-off score is 2.00. b

Fold increase is set as [matches /size in foregound]  [matches/size in background].

28


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Figure 1

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Figure 2

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Figure 3

2



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Figure 4

1


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Figure 5

1



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Figure 6

2


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Figure 7

1



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Graphic for table of contents

2


Systematic Identification and Analysis of Lysine Succinylation in

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