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Proteome-level analysis of metabolism- and stress-related proteins during seed dormancy and germination in Gnetum parvifolium Ermei Chang, Nan Deng, Jin Zhang, Jianfeng Liu, Lanzhen Chen, Xiulian Zhao, M Abbas, Zeping Jiang, and Shengqing Shi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05001 • Publication Date (Web): 28 Feb 2018 Downloaded from http://pubs.acs.org on March 2, 2018
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Proteome-level analysis of metabolism- and stress-related proteins during seed dormancy and germination in Gnetum parvifolium Ermei Chang1*, Nan Deng2*, Jin Zhang1, Jianfeng Liu1, Lanzhen Chen3,4, Xiulian Zhao1, M Abbas1,5, Zeping Jiang1§, Shengqing Shi1§ 1
State Key Laboratory of Tree Genetics and Breeding, Research Institute of Forestry,
Chinese Academy of Forestry, Beijing, China. 2
Institute of Ecology, Hunan Academy of Forestry, Changsha, Hunan, China.
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Institute of Apicultural Research, Chinese Academy of Agricultural Sciences,
Beijing, China. 4
Risk Assessment Laboratory for Bee Products, Quality and Safety of Ministry of
Agriculture, Beijing, China 5
National Engineering Laboratory for Tree Breeding, College of Biological Sciences
and Technology, Beijing Forestry University, Beijing 100083, China. *Co-first author. §
Correspondence:
Dr. Shengqing Shi Chinese Academy of Forestry Research Institute of Forestry State Key Laboratory of Tree Genetics and Breeding
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No. 1 Dongxiaofu, Xiangshan Road Haidian, Beijing 100091, China shi.shengqing@caf.ac.cn
Dr. Zeping Jiang Chinese Academy of Forestry Research Institute of Forestry State Key Laboratory of Tree Genetics and Breeding No. 1 Dongxiaofu, Xiangshan Road Haidian, Beijing 100091, China jiangzp@caf.ac.cn
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ABSTRACT: Gnetum parvifolium is a rich source of materials for traditional
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medicines, food, and oil, but little is known about the mechanism underlying its seed
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dormancy and germination. In this study, we analyzed the proteome-level changes in
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its seeds during germination using isobaric tags for relative and absolute quantitation.
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In total, 1,040 differentially-expressed proteins were identified, and cluster analysis
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revealed the distinct time points during which signal transduction and oxidation–
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reduction activity changed. Gene Ontology analysis showed that “carbohydrate
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metabolic process” and “response to oxidative stress” were the main enriched terms.
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Proteins associated with starch degradation and antioxidant enzymes were important
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for dormancy-release, while proteins associated with energy metabolism and protein
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synthesis were up-regulated during germination. Moreover, protein-interaction
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networks were mainly associated with heat-shock proteins. Furthermore, in accord
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with changes in the energy metabolism-, and antioxidant- related proteins,
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indole-3-acetic acid, POD, and soluble sugar content increased, and the starch content
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decreased in almost all the six stages of dormancy and germination analyzed (S1–S6).
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The activity of superoxide dismutase, abscisic acid and malondialdehyde content
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increased in the dormancy stages (S1-S3), and then decreased in the germination
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stages (S4-S6). Our results provide new insights into G. parvifolium seed dormancy
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and germination at the proteome and physiological levels, with implications for
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improving seed propagation. KEYWORDS: Gnetum parvifolium, proteome, seed germination, ABA, iTRAQ
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Introduction
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The genus Gnetum, along with two other genera (Ephedra and Welwitschia),
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comprise a unique group in the division Gnetophyta 1. The phylogenetic position of
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Gnetophyta is one of the most contentious issues in seed plant systematics. Gnetum is
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a vegetable and nut fruit used for health-care; the leaves are common vegetables and
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spices in Nigeria, Cameroon, Central Africa, Congo, Angola, and Southeast Asia, and
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they are sold to Europe, the USA, and Japan. In Southeast Asia, Gnetum is cultivated
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to produce nuts, and the output of a mature plant reaches 80-100 kg
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Gnetum species reportedly produce many important bioactive compounds (e.g.,
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flavonoids and stilbenoids) that are used in traditional medicines in many countries,
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having hypotensive, antioxidant, anticancer, and antibacterial effects
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their economic value, there has been considerable interest in the introduction,
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domestication, and cultivation of Gnetum species in Africa and Southeast Asia 8. Seed
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propagation is currently one of the main methods of reproducing Gnetum species.
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However, the seeds undergo an after-ripening process, which is followed by a
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prolonged germination period. Thus, there are challenges associated with producing
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Gnetum species in nurseries. In addition, relatively few investigations have attempted
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to characterize seed dormancy and germination in Gnetum.
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The main causes of seed dormancy include the seed coat, hormone levels, embryo
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immaturity, endogenous inhibitors, or the joint action of these factors
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mechanism of seed dormancy-breaking involves many physiological processes,
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ranging from the initiation of internal hormone signals, signal transmission, 4
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. In addition,
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. Because of
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. The
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transcription, protein synthesis, energy metabolism, and the mobilization of storage
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material, to external environmental factors and even cell regeneration
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dormancy-release and germination, seeds enlarge almost solely by cell expansion,
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which requires growth hormones and energy. Carbon metabolic and fermentation
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pathways play important roles affecting different stages of dormancy and germination
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during rice seed germination; Furthermore, inhibitory activity during the early stages
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of dormancy is associated with the production of reactive oxygen species (ROS),
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while antioxidant enzymes regulate the ROS levels during dormancy and germination
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13-15
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stimuli, such as light, with the up- and down-regulation of endogenous
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phytohormones. For example, abscisic acid (ABA) inhibits germination and is
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involved in transcriptional regulation in response to osmotic stress, while
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indole-3-acetic acid (IAA) promotes germination and early seedling development.
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Regardless of this, higher levels of IAA give rise to Pseudomonas isolates and inhibit
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germination as well as negatively regulating the α-amylase activity in durum wheat
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(Triticum turgidum L.) 16.
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Seed dormancy and germination are regulated by many proteins. The emergence and
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development of proteomics technology provide a means to study the mechanisms
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underlying these phenomena. Increasing amounts of data have been generated from
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large-scale analyses of protein expression during dormancy-breaking
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only limited information is available regarding G. parvifolium seeds. Most
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proteome-level studies of germination and development have involved analyses of
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. During
. Both dormancy and germination phenomena are regulated by environmental
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. However,
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model species, such as Arabidopsis thaliana and important crops
using
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two-dimensional electrophoresis, which has been widely used over the past two
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decades. A mass spectrometry-based technique using isobaric tags for relative and
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absolute quantitation (iTRAQ) has been developed for proteome-level investigations
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techniques that achieves sensitive and accurate protein quantification by using
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reporter ion pairs in the low mass range of MS2 spectra 22. Based on iTRAQ data, the
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metabolic activity, regulation of redox homeostasis, and protein expression during
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rice seed germination have been characterized 23. In this study, we set out to analyze
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the proteome-level changes during the germination of G. parvifolium seeds using
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iTRAQ. We also investigated the relationship between changes in hormone content
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and seed dormancy during inhibition of the germination and seed-stratification
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process. Our results provide novel insights into the genetic mechanisms regulating
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seed germination in Gnetum species, and they have implications for dormancy-release
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and enhancing seedling development.
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Methods
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Plant materials and experimental design. G. parvifolium seeds were collected from
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DiaoLuo Mountain in Hainan, China. The seeds were cleaned with water and dried at
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ambient temperature and humidity. River sand was filtered through a sieve with 2-mm
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pores and disinfected with 0.2% KMnO4 prior to use as a layered medium. The
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washed seeds were mixed with wet sand (1:2), and then stratified in pots (20–25°C).
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Seed samples were collected at six time points (stages S1–S6: 0, 30, 60, 120, 150, and
, The iTRAQ system is currently one of the most robust mass-spectrometric
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180 days) (Figure 1A). The samples were frozen in liquid nitrogen and stored at
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−80°C.
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Analysis of physiological changes. Total soluble protein and malondialdehyde
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(MDA) were measured as described by Cui et al.
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superoxide dismutase (SOD) and peroxidase (POD) activity were analyzed using the
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method developed by Wang et al.
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determined as described by Breeze et al. 27. The endogenous hormones IAA and ABA
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were extracted and purified as described by Cui et al.
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estimated using ELISA kits (China Agricultural University, Beijing, China). Three
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independent biological replicates at each stage were acquired for physiological
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analysis. Differences were scored as statistically significant at the P