Proteomic and Metabolomic Analyses of Leaf from Clematis terniflora

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Proteomic and Metabolomic Analyses of Leaf from Clematis terniflora DC. Exposed to High-Level Ultraviolet-B Irradiation with Dark Treatment Bingxian Yang, Xin Wang, Cuixia Gao, Meng Chen, Qijie Guan, Jingkui Tian, and Setsuko Komatsu J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00206 • Publication Date (Web): 20 Jun 2016 Downloaded from http://pubs.acs.org on June 21, 2016

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Journal of Proteome Research

Proteomic and Metabolomic Analyses of Leaf from Clematis terniflora DC. Exposed to High-Level Ultraviolet-B Irradiation with Dark Treatment

Bingxian Yang1,2, Xin Wang2, Cuixia Gao1, Meng Chen1, Qijie Guan1, Jingkui Tian1,*, and Setsuko Komatsu2,*

1 College of Biomedical Engineering & Instrument Science, Zhejiang University, Hangzhou 310027, China 2 National Institute of Crop Science, National Agriculture and Food Research Organization, Tsukuba 305-8518, Japan

*Corresponding Author: Setsuko Komatsu, National Institute of Crop Science, National Agriculture and Food Research Organization, Kannondai 2-1-18, Tsukuba 305-8518, Japan. Tel: +81-29-838-8693. Fax: +81-29-838-8694. E-mail: [email protected] Jingkui Tian, College of Biomedical Engineering & Instrument Science, Zhejiang University, Zheda Road 38, Hangzhou 310027, China. Tel: +86-571-87951301. Fax: +86-571-87951676. E-mail: [email protected]

Running title: Clematis Proteomics under Binary Stress

Abbreviations: UV-B, ultraviolet-B; DPPH, 1,1-diphenyl-2-picrylhydrazyl; LC, liquid chromatography; GC, gas chromatography; TOF, time-of-flight; MS, mass spectrometry; SAM, S-adenosylmethionine; ACC, 1-aminocyclopropane-1-carboxylic acid; GABA, gamma-aminobutyric acid. 1

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ABSTRACT: Clematis terniflora DC. has potential pharmaceutical value; on the other hand, high-level UV-B irradiation with dark treatment led to the accumulation of secondary metabolites. Metabolomic and proteomic analyses of leaf of C. terniflora were performed to investigate the systematic response mechanisms to high-level UV-B irradiation with dark treatment. Metabolites related to carbohydrates, fatty acids, and amino acids, and/or proteins related to stress, cell wall, and amino acid metabolism were gradually increased in response to high-level UV-B irradiation with dark treatment. Based on cluster analysis and mapping of proteins related to amino acid metabolism, the abundances of S-adenosylmethionine synthetase and cysteine synthase as well as 1,1-diphenyl-2-picrylhydrazyl scavenging activity were gradually increased in response to high-level UV-B irradiation with dark treatment. Furthermore, the abundance of dihydrolipoyl dehydrogenase/glutamate dehydrogenase and the content of gamma-aminobutyric acid were also increased following high-level UV-B irradiation with dark treatment. Taken together, these results suggest that high-level UV-B irradiation with dark treatment induces the activation of reactive oxygen species scavenging system and gamma-aminobutyric acid shunt pathway in leaf of C. terniflora.

KEYWORDS: proteomics, metabolomics, Clematis terniflora DC., leaf, ultraviolet-B irradiation, dark treatment

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INTRODUCTION Clematis, which is one of the largest genera in the family Ranunculaceae, contains numerous species with potential pharmaceutical value due to their anti-inflammatory 1-3, antinociceptive, and antipyretic 4 activities. One species in particular, Clematis terniflora DC., which is widely spread throughout Asia, Europe, and Africa 5, has been characterized with respect to the production of alkaloids, saponins, and flavonoids from leaf/stem, root, and flower, respectively 6-8. Although the pharmaceutical effects of root and flower of C. terniflora have not been well studied, the leaf and stem have anti-inflammatory, antinociceptive 9, 10, and anti-tumor 11

effects. These results indicate that the leaf and stem of C. terniflora have a

pharmaceutical potential against disease. Ultraviolet-B (UV-B) is one component of sunlight and is considered to be an environmental stressor for plants 12. Although low-level UV-B irradiation plays a role in regulating plant growth/development 13 and phenolic compounds accumulation 14, exposure to high-level UV-B irradiation leads to the formation of reactive oxygen species 15 and impairs protein synthesis 16. Plants treated with high-level UV-B irradiation with dark treatment also accumulate various secondary metabolites, including Diels-Alder adducts 17, iridoids, caffeoylquinic acids 18, and alkaloids 19. Diels-Alder adducts have anti-tumor activity by inhibiting hypoxia-inducible factor-1 accumulation and vascular endothelial growth factor secretion 20. Iridoids have anti-inflammatory effects through inhibition of the PI3K/Akt signaling pathway 21, whereas alkaloids also have both anti-inflammatory 22 and anti-tumor activities 23. These findings indicate that high-level UV-B irradiation with dark treatment can increase the anti-inflammatory and anti-tumor activities of plants by inducing the accumulation of secondary metabolites. However, the relationship between the 3



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pharmaceutical effects of C. terniflora and high-level UV-B irradiation with dark treatment remains unclear. Metabolomic and proteomic analyses have been used to examine the response mechanisms of plants to UV-B stress 24. Metabolomic analysis revealed that in response to high-level UV-B irradiation, metabolites related to flavonoids, anthocyanins, and polyphenols were increased and that metabolites related to phenolic precursors and steroids were decreased in leaf of Populus x canescens 25. In addition, proteomic analysis indicated that proteins related to the synthesis of caffeoylquinic acids and iridoids were increased in leaf of Lonicera japonica Thunb. exposed to high-level UV-B irradiation with dark treatment 26. However, such treatment leads to a decrease in proteins related to photosynthesis in leaf of Mahonia bealei 27 and Catharanthus roseus 19. These results demonstrate that metabolomic and proteomic approaches are useful for investigating the response mechanisms of plants exposed to UV-B stress. As plants exposed to UV-B stress accumulate a number of pharmaceutically active secondary metabolites 21-23, the treatment of medicinal plants with high-level UV-B irradiation may be a feasible strategy for increasing the production of pharmaceutical compounds. It is therefore important to understand the response mechanisms of plants to UV-B-induced stress, which represents a potential method of medicinal manipulation 28. To explore the response mechanisms of C. terniflora to high-level UV-B irradiation with dark treatment, metabolomic and proteomic analyses of leaf were performed. Furthermore, functional analyses of the identified metabolites and proteins were systemically integrated with the results of bioinformatic and biochemical analyses.

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EXPERIMENTAL PROCEDURES Plant Material and Treatments Seeds of Clematis terniflora DC. were collected and sown in seedbeds after incubation in water. The seedlings were transplanted into potted containers and placed in a greenhouse, which was controlled at 28-30°C, 70%-80% relative humidity, and 160 µmol m-2 sec-1 of white light irradiance. After 6 weeks, the plants were treated without or with UV-B irradiation (275-320 nm) in a cabinet, which was controlled at 25-30°C, 80% relative humidity, and 160 µmol m-2 sec-1 of white light irradiance. The intensity of UV-B irradiation on the surface was 120.8 µW cm-2, which corresponded to high-level UV-B irradiation, was measured using a UV Light Meter (Beijing Normal University, Beijing, China). After UV-B irradiation, plants were incubated in dark or light conditions for 48 h (Figure S1). For morphological analysis, plants were exposed to high-level UV-B irradiation for 5 h and incubated in the dark for an additional 48 h. The number of leaves was counted based on the selection of more than 2 cm of length, the total leaf area was calculated using an automated digital image analysis 29, and the leaf biomass was weighted after drying the material at 70°C for 20 h. For metabolomic and proteomic analyses, plants were exposed to high-level UV-B irradiation for 5 h and incubated in the dark for an additional 48 h. For further proteomic analysis, plants were treated without or with high-level UV-B irradiation for 2 and 5 h, followed by incubation without or with dark for 24 and 48 h. Leaves in the area from the basal 10 to 60 cm of each plant were collected and freeze dried. For morphological, metabolomic, and proteomic analyses, 3, 9, and 3 independent biological replicates were used, respectively.

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Protein Extraction, Purification, and Digestion for Mass Spectrometry Analysis A portion (50 mg) of each freeze-dried leaf sample was added to an acetone solution containing 10% trichloroacetic acid and 0.07% 2-mercaptoethanol 30. The resulting mixture was vortexed, sonicated for 10 min, and then incubated for 1 h at -20°C with vortexing every 15 min. The suspension was centrifuged at 9,000 x g for 20 min at 4°C, and the obtained pellet was washed twice with 0.07% 2-mercaptoethanol in acetone, dried using a Speed-Vac concentrator (Savant Instruments, Hickville, NY, USA), and then resuspended in lysis buffer consisting of 7 M urea, 2 M thiourea, 5% CHAPS, and 2 mM tributylphosphine by vortexing for 1 h at 25°C. The resulting suspension was centrifuged at 20,000 x g for 20 min at 25°C, and the supernatant was collected as crude extract. Protein concentrations were determined using the Bradford assay 31 with bovine serum albumin as the standard. Proteins (100 µg) were enriched with methanol and chloroform to remove any detergent from the sample solutions 32. Briefly, 400 µL methanol was added and mixed with each crude protein extract sample before the further addition of 100 µL chloroform and 300 µL water. After mixing, the samples were centrifuged at 20,000 x g for 10 min to achieve phase separation. The upper aqueous phase was discarded and 300 µL methanol was added slowly to the lower phase. The samples were further centrifuged at 20,000 x g for 10 min, and the obtained pellets were dried, resuspended in 50 mM NH4HCO3, reduced with 50 mM dithiothreitol for 30 min at 56°C, and then alkylated with 50 mM iodoacetamide for 30 min at 37°C in the dark. Alkylated proteins were digested with trypsin and lysyl endopeptidase (Wako, Osaka, Japan) at 1:100 enzyme/protein concentrations at 37°C for 16 h. The resulting tryptic peptides were acidified with formic acid (pH

Proteomic and Metabolomic Analyses of Leaf from Clematis terniflora

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