The high salinity-induced changes of protein profile and the identification of related proteins in Mesembryanthemum crystallinum L.

Abstract

中 文 摘 要 冰花為一耐鹽模式植物，其在幼苗期無法忍受高鹽環境，到成年期時則可適應高鹽環境，且屬於誘導型景天酸代謝植物。本論文主要是探討冰花遭遇鹽逆境時，根部及葉部蛋白質圖譜的變化，且針對負責儲存過多鹽離子的特化細胞-腎型細胞的蛋白圖譜以及細胞間質蛋白圖譜進行分析，並鑑定其主要蛋白質以及鹽誘導蛋白質身份，藉以了解高等植物的耐鹽機制。 首先分析根部及葉部的鹽誘導蛋白質。分別萃取200 mM NaCl處理下不同時間點的總蛋白質進行二維電泳分析，比較蛋白質圖譜在不同時間點下的變化。在根部中，有33個蛋白質點累積量有變化，其中50%為鹽誘導蛋白質，有3個蛋白質點為鹽誘導下新合成蛋白。在葉片中，則有35個蛋白質點累積量有變化，其中60%為鹽誘導蛋白質，有3個蛋白質點為鹽誘導下新合成蛋白。這些鹽誘導蛋白在一般環境下也會持續表現，而在高鹽環境下其累積量增加，推測這些鹽誘導蛋白具有維持細胞基本運作的功能，而在高鹽環境下則可幫助適應逆境。另外在葉部的蛋白質圖譜改變可能和葉片由C3轉變成CAM光合作用有關。 在腎形細胞中，有25個蛋白質點在加鹽處理後有累積量的變化，其中四個蛋白質點累積量增加最為明顯。選取腎形細胞蛋白質主要的蛋白質點以及在鹽逆境下增加最明顯的蛋白質點，經由LC-MS/MS分析，並結合蛋白質N端定序的結果，與蛋白質資料庫進行比對以鑑定蛋白質身份。根據鑑定結果顯示，鹽逆境下腎形細胞蛋白質點累積量增加最明顯的蛋白質為PR-10 protein及phosphoglycerate kinase，推測醣解作用負責提供區隔鈉離子所需的能量。在腎形細胞液泡中的主要蛋白質酶應為cysteine proteinase；而腎形細胞中主要的蛋白質還包含V-type proton-ATPase subunit E，維持質子梯度以提供Na+/ H+ antiporter運送鈉離子進入液泡，以及具有抑制病原菌生長能力的PR-5和PR-10。 另外以冰花癒傷組織為材料，探討鹽逆境下細胞間質蛋白組成變化。結果顯示細胞間質蛋白大部份為鹼性蛋白質，其中20及23 kDa protein在含鹽環境下易釋出。選取細胞間質蛋白質以MALDI-TOF MS分析，並結合蛋白質N端定序的結果，與蛋白質資料庫進行比對以鑑定蛋白質身份。根據鑑定結果細胞間質蛋白質包含fiddlehead-like protein，sodium symporter family protein，chitinase (PR-8 protein)，以及兩種PR-5 proteins。Fiddlehead-like protein以及PR proteins 都具有抗菌的功能，而PR-5 proteins在含鹽環境下易釋出，推測此機制可幫助植物在逆境下抵抗病原菌入侵。 根據以上結果推測，在鹽逆境下腎型細胞中的醣解作用速率增加，以提供較多能量幫助ATPase維持質子梯度，以提供Na+/ H+ antiporter運送鈉離子進入液泡，避免產生離子毒害。此外在鹽逆境下，植物增加PR proteins的累積量，可幫助抵抗病原菌入侵。Abstract Mesembryanthemum crystallinum exhibits model characters for studying inducing response to salt stress. In this study, we focused on the salt-induced changes of protein profile in roots and leaves of M. crystallinum. The epidermal bladder cells, specialized cells of ice plant, are responsible for the sequestration of large amounts of sodium and chloride ions under high salt condition. The main focus of this thesis is to study the salt-induced changes of protein profile in bladder cells and to identify the major proteins of bladder cells that would help to illustrate the salt-tolerant mechanism of ice plant. Total proteins were extracted from 0 or 3-day 200 mM NaCl treated roots or from 0, 3, or 7-day 200 mM NaCl treated leaves and were analyzed by 2-DE. The accumulation of thirty-three protein spots in roots were changed under salt treatment. Among them, 50% were salinity-induced polypeptides and three novel spots were found. The accumulation of thirty-five protein spots in leaves were change under salt treatment. Among them, 60% were salinity-induced polypeptides and three novel spots were found. These proteins were constitutively accumulated under no stress condition and the amount increased under high salinity condition suggesting proteins involved in maintaining basic cellular functions may also play roles in adaptation to salt. The most prominent salt-induced changes is the switches from C3 to CAM photosynthesis and the changes of leaf protein profiles under salt stress are most likely linked to this metabolic transition. The proteins isolated from bladder cells were analyzed by 2-DE gel. The protein profile change of the bladder cells under high salt condition identified 25 spots. Four spots were significantly increased by salt treatment. The identity of the salinity-induced spots and the major spots in bladder cells were identified by LC-MS/MS and N-terminal sequences. We successfully identified ten abundant proteins from bladder cells protein extracts. The major spots were cysteine proteinase, V-type proton-ATPase subunit E, PR-5 and PR-10. The salinity-induced spots were phosphoglycerate kinase and PR10. The result showed the major protease in vacuole of bladder cells was cysteine proteinase. The accumulation of PR proteins in bladder cells would help to resist pathogens infection. The V-type proton-ATPase established the proton gradient to uptake sodium through the Na+/ H+ antiporter. The changes of intercellular protein under salt stress would mimic the effect of salt stress during the secretion process in intact plants. Two polypeptide in 20 kDa and 23 kDa were showed up in the salt containing buffer. The identity of the salinity-induced bands and the major bands of intercellular proteins were identified by MALDI-TOF MS and N-terminal sequences. The identifications of intercellular protein were fiddlehead-like protein, sodium symporter family protein, chitinase (PR-8), and two PR-5 proteins. The PR-5 proteins were only showed under the extraction buffer containing salt suggesting the PR proteins were quickly released to help defense the pathogens infection when external environment encountered high salinity. This study showed induction of vacuolar H+-ATPase and the glycolytic enzymes under high salinity condition. The results indicate efficient generation of H+ gradient is an important factor for salt tolerance in this halophyte. In addition to maintain ion homeostasis, epidermal bladder cells also act as the first line of defense against pathogens. The accumulation of PR proteins in extracellular space of plants or cells provides an active defense mechanism for pathogens.Abstract………………………………………………………………………………..I Abstract of Chinese………………………………………………………………….III Catalog……………………………………………………………………….. Ⅳ Catalog of tables……………………………………………………………………VI Catalog of figures……………………………………………………………………. VII Introduction…………………………………………………………….………………..1 Soil salinity…………….…………………………………………………………..1 Mesembryanthemum crystallinum L……………………..…..……………………2 The application of proteomics……..…………………………...………………….2 Pathogenesis-related protein family…………….…………………………………6 Materials and methods………………….…………………………………………….11 Material.…….…………………………………………………………………….11 (A) Growth of plants……………...………………………………………...11 (B) Callus culture……………………...……………………………………12 Methods…………………………………………….…………………………….13 (A) Protein extraction……………..…………….………………………….13 a. Total protein extraction …………………...……………………….13 b. Bladder cell protein extraction………………………………..……13 c. Intercellular protein extraction (A)………………..…………...…..13 d. Intercellular protein extraction (B)…………….…………………..14 e. Phenol extraction of total soluble protein…………………………14 (B) Electrophoresis…………………………………………………………15 a. PAGE……………………………………………………..…..….15 I SDS-PAGE…………………………………………...……….15 II Tricine SDS-PAGE…………………………………………..16 b. Two-Dimensional Electrophoresis….……………………………...17 I Isoelectric focusing………………………….………………..17 i Sample directly rehydration………………………….17 ii Apply sample after gel rehydration…….…………….17 iii Ettan IPGphor cup loading……………..…………….18 II Second dimension……………………………………………18 (C) Western blot……….……………………………………………….....18 (D) In gel trypsin digestion…………..…………………………….……….19 (E) N-terminal amino acid sequence sample prepare…………..……..……20 (F) Protein identification MALDI-TOF………..………………………....21 (G) Protein identification……..……………………………………….…....22 (H) Yeast growth assay……………………………………………………..22 Result……………………...………………………………………………………..…..24 (A) High salinity-induced changes of protein profile in roots……...………………..24 (B) High salinity-induced changes of protein profile in leaves……………………...25 (C) Protein profile in epidermal bladder cells……………………………………...25 (D) Protein profile of bladder cells under salt stress……………………………...….27 (E) Protein profile of intercellular protein…………………………………...………28 (F) Identify basic intercellular proteins by high salt extraction…………………...…29 (G) Comparison of three IEF separation methods….……………..………………….30 Discussion ………………………………………………………………………..…….32 The high salinity-induced proteins in roots of ice plant……………..…………………32 The high salinity-induced protein in leaves of ice plant……………………………....32 The function of the identified proteins in bladder cells ……………..………...…….....33 The function of PR proteins under high salt treatment in ice plant…………………….36 E.Referance………………………………………………………..……………………39 F.Appendix……………………………………………………………………………7