Characterization of bladder cell-specific mcSKD1 and its interacting proteins in halophyte Mesembryanthemum crystallinum L.

冰花(Mesembryanthemum crystallinum L.)是一種具有C3-CAM光合作用轉換形式植物可生存於高鹽環境的耐鹽模式植物，表皮特化的腎型細胞在高鹽環境下液胞會迅速膨大，具有儲存水分及區隔過量鈉離子的功能。利用酸性下會呈紅色的染劑neutral red浸染不同時期的冰花表皮細胞，觀察到冰花腎型細胞液胞pH值除了會隨著發育逐漸由酸性轉變為中性外，幼年期的腎型細胞在照光時液胞pH值亦會快速升高，顯示腎型細胞的代謝旺盛並非僅供儲存之用，還具有調節離子平衡之功能。冰花的mcSKD1 (suppressor of K+ transport growth defect)是一個會在冰花的腎型細胞表現的鹽誘導基因，mcSDK1蛋白與酵母菌的液胞運送相關蛋白VPS4 (vacuolar protein sorting 4)和阿拉伯芥AAA-type ATPase (ATPase associated with a variety of cellular activities)具有高度的相似性，並觀察到mcSKD1主要分布在冰花細胞質之ER/Golgi network。為了更進一步瞭解mcSKD1蛋白之功能，以酵母菌雙雜交系統分別由阿拉伯芥基因庫中篩選出與SKD1具有蛋白質交互作用的過氧化逆境相關的catalase 3、細胞壁結構的AGP21 (arabinogalactan protein 21)等蛋白。以及由冰花根部加鹽處理三天所製成的基因庫篩選出與SKD1具有蛋白質交互作用蛋白mcSNF1 (sucrose non-fermenting 1)及mcCPN1 (copine 1)等蛋白。SNF1已知為一參與醣類代謝及逆境相關訊息傳遞作用之蛋白激酶，mcSNF1還具有UBA (ubiquitin associated) domain，mcCPN1蛋白具有與細胞膜蛋白質運送相關之VWA domain及具有E3 ubiquitin ligase活性之RING domain，mcSNF1與mcCPN1也會受加鹽誘導提高蛋白累積量。免疫共沈澱證明了mcSKD1與mcSNF1與mcCPN1具有蛋白質交互作用，以pull-down的蛋白產物比例計算發現，加鹽處理會使mcSKD1與mcSNF1結合的比例提高三倍。利用蔗糖梯度分離microsome蛋白以及細胞免疫染色法，發現加鹽處理會快速改變mcSKD1、mcSNF1和mcCPN1在細胞中分佈的位置，且colocolization的比例提高。當以破壞細胞骨架的藥劑處理細胞時，mcSKD1及mcSNF1在細胞中的分布改變，累積在細胞邊緣，推測細胞骨架參與mcSKD1在細胞中的運輸。鹽處理會造成阿拉伯芥snf1或copine1突變株中atSKD1不正常的聚集，並對鹽更加敏感。實驗結果推測冰花mcSKD1蛋白透過與逆境訊息傳遞mcSNF1、細胞膜相關蛋白mcCPN1的交互作用，參與鹽逆境時ubiquitin相關的蛋白質運送或是逆境訊息傳遞，幫助耐鹽植物冰花在高鹽環境下維持正常生理代謝。The halophyte Mesembryanthemum crystallinum L. (ice plant) is an inducible CAM and a model plant for studying salt-tolerant mechanisms in higher plants. It contains specialized epidermal bladder cells (EBCs) which rapidly expend under salt stress. The major functions of EBCs are maintaining ion homeostasis and water storage. During plant development, increase in vacuolar pH was observed in EBCs. In addition, light-induced rapid change in vacuolar pH was found in juvenile stage of ice plant indicating EBCs are metabolic active cells. A salt-induced gene mcSKD1 (suppressor of K+ transport growth defect) is highly expressed in EBCs. It has high homology to yeast VPS4 (vacuolar protein sorting 4) and Arabidopsis AAA-type ATPase (ATPase associated with a variety of cellular activities). Immunofluorescence labeling showed mcSKD1 protein was located in cytoplasm around ER/Golgi network. Yeast two-hybrid screen was performed to identify mcSKD1-interacting proteins. Using a library constructed from Arabidopsis, catalase 3 and AGP21 (arabinogalactan protein 21) were identified. Using a library constructed from salt-treated ice plant roots, mcSNF1 (sucrose non-fermenting 1) and mcCPN1 (copine 1) were further characterized. Yeast SNF1 is a ser/thr kinase that plays an important role in carbon metabolism and stress signaling. The SNF1 protein identified in ice plant contained an extra UBA (ubiquitin associated) domain. Sequence analysis of mcCPN1 showed it contains a VWA domain for membrane trafficking and a RING domain for protein ubiquitination. The accumulation of mcSNF1 and mcCPN1 was both induced by salt stress. Co-immunoprecipitation experiment showed mcSKD1 interacted with both mcSNF1 and mcCPN1 in vitro. In vivo pull-down assay showed a 3-fold increase in the association between mcSKD1 and mcSNF1 under salt stress. Microsomal fractionation and immunolabeling experiments showed salt induced rapid changes in cellular localization of mcSKD1, mcSNF1, and mcCPN1. When the cytoskeleton was disrupted, the distribution of mcSKD1 and mcSNF1 was altered, as seen by abnormal aggregation around plasma membrane. The result suggested that mcSKD1 is trafficking along the cytoskeleton. Arabidopsis snf1 and copine 1 mutants showed more salt sensitive than the wild type and aggregation of atSKD1 inside mutant cells. The results suggested that mcSNF1 and mcCPN1 function together with mcSKD1 in ubiquitin-related protein trafficking and stress signal transduction in order to maintain normal growth under high salinity.Catalog: Abstract in Chinese II Abstract IV Catalog VI Catalog of tables VIII Catalog of figures IX Preface 1 Chapter 1 6 Abstract 7 Introduction 8 Material and methods 10 Results 12 Discussion 14 References 17 Chapter 2 24 Introduction 25 Material and methods 30 Results 34 Discussion 39 References 45 Chapter 3 73 Introduction 74 Material and methods 78 Results 84 Discussion 91 References 95 Catalog of tables: Chapter 2 Table 1. The category “tissue” was used in similarity search. Following genes are sorted with AtSKD1…………………………………………………. 54 Table 2. The category “nutrient treatment” was used in similarity search. Following genes are sorted with AtSKD1………………………………… 55 Table 3. The category “abiotic stress” was used in similarity search. Following genes are sorted with AtSKD1…………………………………………….. 56 Table 4. Putative mcSKD1-interacting proteins identified from Arabidopsis library…………………………………………………………………….. 57 Table 5. Putative mcSKD1-interacting proteins identified from salt-stressed ice plant library……………………………………………………………….. 58 Chapter 3 Table 1. Search mcSKD1 protein modification pattern on PROSITE database……………………………………………………………….. 102 Table 2. Search mcSNF1 protein modification pattern on PROSITE database………………………………………………………………... 103 Table 1. Search mcCPN1 protein modification pattern on PROSITE database………………………………………………………………... 104 Catalog of figures: Chapter 1 Fig. 1. Morphology and vacuole acidity of ice plant EBC during development... 21 Fig 2. The light-induced decrease in acidity of EBCs…………………………... 22 Fig 3. Change in composition of raphide crystals……………………………….. 23 Chapter 2 Fig 1. Quantitative analysis of atSKD1 expression under various stresses……... 60 Fig 2. Total cellular protein extracted from 10-day-old cultures ice plant cells… 61 Fig 3. Distribution of mcSKD1 in microsomal fractions………………………... 62 Fig 4. Double-labeling immunofluorescence……………………………………. 63 Fig 5. Immunstaining of ice plant leaf EBCs……………………………………. 64 Fig 6. The accumulation of atSKD1 in 8-week-old Arabidopsis plants…………. 65 Fig 7. The localization of atSKD1 in cultured Arabidopsis cells………………... 66 Fig 8. Isolation of Poly A+ mRNA for yeast two hybrid screening library……… 67 Fig 9. In vivo interaction between mcSKD1 and Catalase 3 or AGP21………… 68 Fig 10. In vivo interaction between mcSKD1 and Copine 1 or SNF-1…………. 69 Fig 11. Arabidopsis QTL markers showing positions of SKD1-interaction proteins…………………………………………………………………… 70 Fig 12. Characterization of the mcSNF1 from ice plant………………………… 71 Fig 13. Characterization of the mcCPN1 from ice plant………………………… 72 Chapter 3 Fig 1. Time course expression of mcSNF1in plant callus under salt stress……... 105 Fig 2. Time course expression of mcCPN1 under salt stress…………………… 106 Fig 3. In vitro coimmunoprecipitation of mcSKD1 with mcCPN1 or mcSNF1… 107 Fig 4. The salt stress changed protein-protein interaction ratio of mcSKD1, mcSNF1 and mcCPN1.…………………………………………………… 108 Fig 5. Double-labeling immunofluorescence of mcSKD1 and mcCPN1 in ice plant cells.………………………………………………………………… 109 Fig 6. Double-labeling immunofluorescence of mcSKD1 and mcSNF1 in ice plant cells………………………………………………………………… 110 Fig 7. Time-course accumulation of mcSKD1, mcSNF1 and mcCPN1 within 24 hours of salt stress……………………………………………………….. 111 Fig 8. mcSNF1 is a extracellular protein.………………………………………. 112 Fig 9. Analysis of protein modification…………………………………………. 113 Fig 10. Distribution of mcSKD1, mcSNF1 and mcCPN1 in microsomal fractions of ice plant leaves.……………………………………………… 114 Fig 11. Distribution of mcSKD1, mcSNF1, and mcCPN1 in microsomal fractions.………………………………………………………… 115 Fig 12. Cell viability test………………………………………………………… 116 Fig 13. Initial changes in cellular distribution of mcSKD1, mcSNF1, and mcCPN1 by 200 mM NaCl.………………………………………………. 117 Fig 14. The effects of cytoskeleton-disrupting drugs on mcSDK1 distribution and endocytosis pathway in ice plant cells.……………………………. 118 Fig 15. The effects of cytoskeleton-disrupting drugs on mcSKD1, mcSNF1 and mcCPN1 localization……………………………………………………………. 119 Fig 16. Accumulation of atSKD1 protein in wild type and akin10, copine1 mutants.………………………………………………………………… 120 Figure 17. Dose-dependent accumulation of atSKD1 protein in wild type Arabidopsis and copine1, snf1 mutants.………………………………….. 121 Figure 18. Double-labeling immunofluorescence on wild type Arabidopsis, snf1, and copine1 mutants……………………………………………. 12

Analysis of K+/Na+ ion homeostasis and related gene expression in NaCl stress environments.

當植物受到鹽逆境時，會因為細胞內累積過多的氯化鈉離子，而抑制細胞質酵素活性並造成細胞內外離子的不平衡，影響鉀離子的吸收，進而引發缺鉀及缺水等傷害。為探討植物細胞對鹽逆境下對鉀離子吸收之影響，本篇論文以耐鹽植物冰花(Mesembryanthemum crystallinum L.)及不耐鹽植物阿拉伯芥(Arabidopsis thaliana)之培養細胞為材料，施予鹽逆境並配合不同濃度之鉀離子處理，測量細胞生長狀況及細胞內鉀鈉離子的含量及比值，以評估不同植物間對於鹽逆境下離子平衡的差異性。由細胞在不同鈉鉀離子濃度的培養基中生長的狀況可以發現，雖然冰花細胞所能承受的鹽逆境高於阿拉伯芥細胞，但無論是冰花或是阿拉伯芥細胞，培養在低鉀的環境中會使對鹽逆境的敏感度上升，造成過高的鈉鉀比值。當細胞培養在以氯化鉀造成高鉀環境的培養基時，細胞生長會受到抑制，以非滲透性溶質mannitol或硫酸鉀取代氯化鉀之培養基，會改善生長抑制的情況，推測過高濃度之氯離子會抑制細胞生長。雖然高鉀環境會限制細胞之生長，但是冰花及阿拉伯芥細胞在高鉀高鹽培養初期，都可以藉由培養基所提供的高濃度鉀離子維持較低之鈉鉀比值。 除了評估冰花細胞生長及鈉鉀離子含量外，並利用一鉀離子運輸相關mcSKD1基因，觀察基因表現及細胞離子平衡的關係。由北方墨點法分析結果得知，mcSKD1基因在鹽逆境及缺鉀逆境初期表現量會下降，之後表現量會增加。配合之前的生理研究結果發現，鹽逆境下mcSKD1基因表現，可降低冰花培養細胞中鈉鉀比值，推測此基因參與鈉鉀離子平衡之機制。由mcSKD1基因在組織專一性的表現得知，在未受鹽逆境的環境下，mcSKD1在發育中的子房及根尖的表現，推測mcSKD1基因參與提供新生組織營養的功能。鹽逆境下mcSKD1基因會在根部的表皮、莖部的皮層以及葉部腎型細胞表現，與區隔鈉離子的作用相關；由冰花葉片的鉀鈉元素分析結果得知，當mcSKD1 基因在根部表現時，可提高葉片中鉀離子的含量，表現量不足時，葉片中鉀離子量逐漸下降，推測mcSKD1基因參與冰花耐鹽機制中維持細胞鉀離子平衡的途徑。When plants under high salinity stress, excess NaCl ions accumulate inside the cells, and as the results, the activities of cytosolic enzymes are inhibited and ion homeostasis is interfered. Moreover, the uptake of essential element potassium is severely affected. To examine the effects of high salt on the uptake of potassium, we used well-defined culture media to culture suspension cells of halophyte Mesembryanthemum crystallinum (ice plant) and glycophyte Arabidopsis thaliana. The growth rates and the contents of Na+ and K+ were measured in cells grown in different combinations of Na+ and K+ concentrations. Although ice plant had higher ability to grow in salt-containing media, yet the sensitivity to high salt increased as the concentration of K+ decreased in the culture media in both plant cells. High KCl-containing media inhibited the growth rate of both cells and replacement of KCl by mannitol or K2SO4 would alleviate the inhibitory effect suggesting that high concentration of Cl- in the culture media caused this growth inhibition. At the initial stage of salt stress, both cells maintained lower sodium/potassium ratios when a high external K+ was supplied. In addition to the growth and ion content measurements, the expressions of an ice plant gene mcSKD1 that has been related to potassium uptake were also examined. As the results from Northern blotting, the expression of mcSKD1 decreased at the initial stage of high salt or low potassium treatments. The levels of mcSKD1 transcript increased as the stress persisted. The increase of mcSKD1 expression parallels to the decrease of cellular Na+/K+ ratio suggesting that this gene be involved in the regulation of ionic balance in ice plant under salt stress. Tissue-specific expression found mcSKD1 gene was expressed in the developing ovary and root tips of the unstressed plants suggesting this gene is involved in nourishing the young developing tissues. Under salt stress, the expressions of mcSKD1 were found in tissues that are responsible for the compartmentation of excess Na+, such as the epidermal cells of roots, the cortex of stems and the specialized epidermal bladder cells of leaves. Together with the analyses of sodium and potassium ion contents, we showed the expression of mcSKD1 in the roots was parallel to the increase of potassium concentrations in the leaves. The function of mcSKD1 protein may play a part in maintaining the potassium homeostasis in salt-stressed ice plant, an important pathway contributing to the overall mechanism of salt adaptation in this halophyte.中文摘要………………………………………………………..…………………I 英文摘要………………………………………………………….………………II 壹、 前言：………………………………………………………………………. 1 一、 耐鹽植物冰花……………………………………………………..2 二、 鹽逆境下阿拉伯芥的反應………………………………………..3 三、 鹽逆境下鉀離子在植物細胞內離子平衡上所扮演的角色……..4 四、 冰花耐鹽相關基因mcSKD1………….…………………………..6 貳、 材料與方法:………………………………………………………………….8 一、 實驗材料：…………………………………………………………8 （一） 冰花盆栽……………………………………………………..8 （二） 細胞培養……………………………………………………..8 二、 實驗方法：………………………………………………………..9 （一） 植物體中Na, K, Ca, Mg之測定…………………………….9 （二） Total RNA之萃取…………………………………………..10 （三） 北方墨點法分析……………………………………………11 （四） 利用RT-PCR方法擴增全長SKD1基因…………………..12 （五） PCR產物之選殖……………………………………………13 （六） In situ RT-PCR and In situ hybridization…………………...14 （七） 掃瞄式電子顯微鏡觀察冰花葉表腎型細胞………………18 參、 結果…………………………………………………………………20 肆、 討論……………………………………………………………………29 伍、 參考文獻………………………………………………………………3