Proteome-level analysis of metabolism- and stress-related proteins

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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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Journal of Agricultural and Food Chemistry

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.

3

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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ACS Paragon Plus Environment


No. 1 Dongxiaofu, Xiangshan Road Haidian, Beijing 100091, China [email protected]

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 [email protected]

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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


2,3

. In addition,

4-7

. Because of

9-11

. 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

5


17

. However,


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17-20

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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

Proteome-level analysis of metabolism- and stress-related proteins

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