and stress-related proteins during seed dormancy and germination in

Subscriber access provided by Kaohsiung Medical University

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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.

Page 1 of 41

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

1

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]

2


Page 2 of 41

Page 3 of 41


1

ABSTRACT: Gnetum parvifolium is a rich source of materials for traditional

2

medicines, food, and oil, but little is known about the mechanism underlying its seed

3

dormancy and germination. In this study, we analyzed the proteome-level changes in

4

its seeds during germination using isobaric tags for relative and absolute quantitation.

5

In total, 1,040 differentially-expressed proteins were identified, and cluster analysis

6

revealed the distinct time points during which signal transduction and oxidation–

7

reduction activity changed. Gene Ontology analysis showed that “carbohydrate

8

metabolic process” and “response to oxidative stress” were the main enriched terms.

9

Proteins associated with starch degradation and antioxidant enzymes were important

10

for dormancy-release, while proteins associated with energy metabolism and protein

11

synthesis were up-regulated during germination. Moreover, protein-interaction

12

networks were mainly associated with heat-shock proteins. Furthermore, in accord

13

with changes in the energy metabolism-, and antioxidant- related proteins,

14

indole-3-acetic acid, POD, and soluble sugar content increased, and the starch content

15

decreased in almost all the six stages of dormancy and germination analyzed (S1–S6).

16

The activity of superoxide dismutase, abscisic acid and malondialdehyde content

17

increased in the dormancy stages (S1-S3), and then decreased in the germination

18

stages (S4-S6). Our results provide new insights into G. parvifolium seed dormancy

19

and germination at the proteome and physiological levels, with implications for

20

improving seed propagation. KEYWORDS: Gnetum parvifolium, proteome, seed germination, ABA, iTRAQ

21 3



Page 4 of 41

22

Introduction

23

The genus Gnetum, along with two other genera (Ephedra and Welwitschia),

24

comprise a unique group in the division Gnetophyta 1. The phylogenetic position of

25

Gnetophyta is one of the most contentious issues in seed plant systematics. Gnetum is

26

a vegetable and nut fruit used for health-care; the leaves are common vegetables and

27

spices in Nigeria, Cameroon, Central Africa, Congo, Angola, and Southeast Asia, and

28

they are sold to Europe, the USA, and Japan. In Southeast Asia, Gnetum is cultivated

29

to produce nuts, and the output of a mature plant reaches 80-100 kg

30

Gnetum species reportedly produce many important bioactive compounds (e.g.,

31

flavonoids and stilbenoids) that are used in traditional medicines in many countries,

32

having hypotensive, antioxidant, anticancer, and antibacterial effects

33

their economic value, there has been considerable interest in the introduction,

34

domestication, and cultivation of Gnetum species in Africa and Southeast Asia 8. Seed

35

propagation is currently one of the main methods of reproducing Gnetum species.

36

However, the seeds undergo an after-ripening process, which is followed by a

37

prolonged germination period. Thus, there are challenges associated with producing

38

Gnetum species in nurseries. In addition, relatively few investigations have attempted

39

to characterize seed dormancy and germination in Gnetum.

40

The main causes of seed dormancy include the seed coat, hormone levels, embryo

41

immaturity, endogenous inhibitors, or the joint action of these factors

42

mechanism of seed dormancy-breaking involves many physiological processes,

43

ranging from the initiation of internal hormone signals, signal transmission, 4


2,3

. In addition,

4-7

. Because of

9-11

. The

Page 5 of 41


44

transcription, protein synthesis, energy metabolism, and the mobilization of storage

45

material, to external environmental factors and even cell regeneration

46

dormancy-release and germination, seeds enlarge almost solely by cell expansion,

47

which requires growth hormones and energy. Carbon metabolic and fermentation

48

pathways play important roles affecting different stages of dormancy and germination

49

during rice seed germination; Furthermore, inhibitory activity during the early stages

50

of dormancy is associated with the production of reactive oxygen species (ROS),

51

while antioxidant enzymes regulate the ROS levels during dormancy and germination

52

13-15

53

stimuli, such as light, with the up- and down-regulation of endogenous

54

phytohormones. For example, abscisic acid (ABA) inhibits germination and is

55

involved in transcriptional regulation in response to osmotic stress, while

56

indole-3-acetic acid (IAA) promotes germination and early seedling development.

57

Regardless of this, higher levels of IAA give rise to Pseudomonas isolates and inhibit

58

germination as well as negatively regulating the α-amylase activity in durum wheat

59

(Triticum turgidum L.) 16.

60

Seed dormancy and germination are regulated by many proteins. The emergence and

61

development of proteomics technology provide a means to study the mechanisms

62

underlying these phenomena. Increasing amounts of data have been generated from

63

large-scale analyses of protein expression during dormancy-breaking

64

only limited information is available regarding G. parvifolium seeds. Most

65

proteome-level studies of germination and development have involved analyses of

12

. During

. Both dormancy and germination phenomena are regulated by environmental

5


17

. However,


Page 6 of 41

17-20

66

model species, such as Arabidopsis thaliana and important crops

using

67

two-dimensional electrophoresis, which has been widely used over the past two

68

decades. A mass spectrometry-based technique using isobaric tags for relative and

69

absolute quantitation (iTRAQ) has been developed for proteome-level investigations

70

21

71

techniques that achieves sensitive and accurate protein quantification by using

72

reporter ion pairs in the low mass range of MS2 spectra 22. Based on iTRAQ data, the

73

metabolic activity, regulation of redox homeostasis, and protein expression during

74

rice seed germination have been characterized 23. In this study, we set out to analyze

75

the proteome-level changes during the germination of G. parvifolium seeds using

76

iTRAQ. We also investigated the relationship between changes in hormone content

77

and seed dormancy during inhibition of the germination and seed-stratification

78

process. Our results provide novel insights into the genetic mechanisms regulating

79

seed germination in Gnetum species, and they have implications for dormancy-release

80

and enhancing seedling development.

81

Methods

82

Plant materials and experimental design. G. parvifolium seeds were collected from

83

DiaoLuo Mountain in Hainan, China. The seeds were cleaned with water and dried at

84

ambient temperature and humidity. River sand was filtered through a sieve with 2-mm

85

pores and disinfected with 0.2% KMnO4 prior to use as a layered medium. The

86

washed seeds were mixed with wet sand (1:2), and then stratified in pots (20–25°C).

87

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

6


Page 7 of 41


88

180 days) (Figure 1A). The samples were frozen in liquid nitrogen and stored at

89

−80°C.

90

Analysis of physiological changes. Total soluble protein and malondialdehyde

91

(MDA) were measured as described by Cui et al.

92

superoxide dismutase (SOD) and peroxidase (POD) activity were analyzed using the

93

method developed by Wang et al.

94

determined as described by Breeze et al. 27. The endogenous hormones IAA and ABA

95

were extracted and purified as described by Cui et al.

96

estimated using ELISA kits (China Agricultural University, Beijing, China). Three

97

independent biological replicates at each stage were acquired for physiological

98

analysis. Differences were scored as statistically significant at the P

and stress-related proteins during seed dormancy and germination in

Recommend Documents