中 文 摘 要
蛋白質酪胺酸磷酸化在動物細胞中已有廣泛的研究且已知在動物細胞的程式性死亡中扮演一個重要的角色。然而, 酪胺酸磷酸化的現象在植物細胞的程式性死亡過程中仍屬未知。此實驗證實了D-甘露醣在水稻的懸浮細胞中會引起水稻懸浮細胞的死亡以及DNA的斷裂。水稻懸浮細胞以D-甘露糖處理24小時後, 兩個蛋白質分子20 kDa與43 kDa酪胺酸磷酸化的現象明顯上升。水稻懸浮細胞在D-甘露糖中經過3天的處理後, 兩個蛋白質分子16 kDa與20 kDa酪胺酸磷酸化的現象明顯降低。此外, 在老化的水稻懸浮細胞中也發現有DNA斷裂的現象以及兩個蛋白質分子26kDa與40kDa酪胺酸磷酸化的現象。同時, 在D-甘露糖處理過程中也發現兩個MAPKK (mitogen-activated rotein kinase kinase) (MEK) /MAPK訊息傳導途徑的基因OsMEK以及 OsMAPK2 的表現量明顯增加。本實驗證明酪胺酸磷酸化改變的現象以及(MEK)/MAPK訊息的傳導和水稻細胞的死亡有關。

ABSTRCT
In mammals protein tyrosine phosphorylation plays an important role in the activation of programmed cell death. However, tyrosine phosphorylation involved in cell death has not been examined in plants. These studies demonstrated that D-mannose induced cell death and DNA fragmentation in rice suspension cells. In the presence of mannose for 24 hours, tyrosine phosphorylation of two proteins, 20 kDa and 43 kDa markedly increased. After incubating 3 days, the level of phosphotyrosine accumulation declined in bands of 16 and 20 kDa. In addition, the occurrence of DNA fragmentation and two tyrosine-phosphorylated proteins, 26 kDa and 40 kDa, were detected in aged suspension-cultured cells. The expression of genes that encode mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK signalling pathway, OsMEK and OsMAPK2, are up regulated during D-mannose treatment. The results provide evidence that protein tyrosine phosphorylation as well as MEK/MAPK signalling pathway is associated with cell death in rice.

CONTENTS

ACKNOWLEDGEMENTS…………………………………………………….…….1
CHINESE ABSTRACT………………………………………………………………2
ABSTRACT…………………………………………………………………………..3
CONTENTS……………………………………………………………………..……4
LIST OF TABLE………………………………………………….….……………….6
LIST OF FIGURES………………………………………………………….….…….7
ABBREVIATION……………………………………………………………….…....9
I. INTRODUCTION…………………………………………………………………10
1. Programmed cell death……………………………………………………..….11
1.1 Morphological characteristics of programmed cell death…………………..11
1.2 Biochemical hallmark of programmed cell death…………………………12
2. Programmed cell death in plants……………………………………………….13
3. Cytochrome C mediates PCD………………………………………………….14
3.1 Cytochrome c induce caspase activation…………………………………..14
3.2 Involvement of caspase-like proteases in plants……………………………15
4. Tyrosine phosphorylation in programmed cell death…………………………...15
4.1 Plant protein kinase and signal transduction……………………………….15
4.2 Protein tyrosine phosphorylation regulates apoptosis………………………16
5. The MEK/MAPK pathway in the regulation of PCD…………………………..17
5.1 The MAP kinase pathways………………………………………………...17
5.2 Involvement of a MAPK signalling pathways in apoptosis………….……18
6. The effect of D-mannose on plant metabolism………………………………..19
7. The purpose of this study………………………………………………………20
Ⅱ. MATERIALS AND METHODS…………………………………………….……21
1. Plant Material and Treatment…………………………………………………...21
2. Viability Staining………………………………………………………………..21
3. Fresh Weight and Dry Weight Determination……………………………….….21
4. TUNEL Assay…………………………………………………………………..21
5. DNA Ladder Analysis…………………………………………………………..22
6. Preparation of Protein Extracts………………………………………………….23
7. Electrophoresis and Western Blot Analysis…………………………………23
Ⅲ.RESULTS……………………………………………….25
1. D-mannose induces cell death in rice cells……………………………………..25
2. Detection of DNA fragmentation in D-mannose-treated rice cells…….………25
3. Changes in protein tyrosine phosphorylation during D-mannose
induced cell death……………………………………………………………….26
4. DNA fragmentation is associated with aging…………………………………..27
5. Tyrosine phosphorylation of two proteins, 26kDa and 40kDa, occurs in
aged rice suspension-cultured rice cells…………………………………………27
Ⅳ.DISCUSSION…………………………………………….28
Ⅴ. REFERENCES…………………………………………………32
Ⅵ. APPENDIX
Involvement of a MAPK signalling pathway in D-mannose treated
cultures……………………………………………………………………………….50

REFERENCES

Afanas’ev VN, Korol’ BA, Mantsygin YA, Nelipovich PA, Pechatnikov VA, Umansky SR. 1986. Flow cytometry and biochemical analysis of DNA degradation characteristic of two types of cell death. FEBS Letters 194, 347-350.

Anderson P. 1997. Kinase cascades regulating entry into apoptosis. Microbiology and Molecular Biology Review 61, 33-46.

Arends MJ, Morris RG, Wyllie AH. 1990. Apoptosis. The role of the endonuclease. American Journal of Pathology 136, 593-608.

Asai T, Stone JM, Heard JE, Kovtun Y, and Yorgey P. 2000. Fumonisin B1-induced cell death in Arabidopsis protoplasts requires jasmonate-, ethylene-, and salicylate-dependent signaling pathways. The Plant Cell 12, 823-1835.

Balk J, Leaver CJ, McCabe PF. 1999. Translocation of cytochrome c from the mitochondria to the cytosol occurs during heat-induced programmed cell death in cucumber plants. FEBS Letters 463, 151-154.

Barizza E, Schiavo FL, Terzi M, Filippini F. 1999. Evidence suggesting protein tyrosine phosphorylation in plants depends on the developmental conditions. FEBS Letters 447, 191-194.

Bethke PC, Lonsdale JE, Fath A, Jones RL. 1999. Hormonally regulated programmed cell death in barley aleurone cells. The Plant Cell 11, 1033-1045.

Bonvin C, Guillon A, Bemmelen MXV, Gerwins P, Johnson GL, Widmann C. 2002. Role of the amino-terminal domains of MEKKs in the activation of NF kappa B and MAPK pathways and in the regulation of cell proliferation and apoptosis. Cellular Signalling 14,123-131.

Callard D, Axelos M, Mazzolini L. 1996. Novel molecular markers for late phases of the growth cycle of Arabidopsis thaliana cell-suspension cultures are expressed during organ senescence. The Plant Physiology 112, 705-715.

Chen YR, Meyer CF, Tan TH. 1996. Persistent activation of c-Jun N-terminal kinase 1 (JNK1) in radiation-induced apoptosis. The Journal of Biological Chemistry 271, 631-634.

Cross TG, Scheel-Toellner D, Henriquez NV, Deacon E. 2000. Serine/Threonine protein kinases and apoptosis. Experimental Cell Research 256, 34-41.

Dai Y, Yu C, Singh V, Tang L, Wang Z, McInistry R, Dent P, Grant S. 2001. Pharmacological inhibitors of the mitogen-activated protein kinase (MAPK) kinase/MAPK cascade interact synergistically with UCN-01 to induce mitochondrial dysfunction and apoptosis in human leukemia cells. Cancer Research 61,5106-5115.

Daugas E, Nochy D, Ravagnan L, Loeffler M, Susin SA, Zamzami N, Kroemer G. 2000. Apoptosis-inducing factor (AIF): a ubiquitous mitochondrial oxidoreductase involved in apoptosis. FEBS Letters 476, 118-123.

Earnshaw WC. 1995. Nuclear changes in apoptosis. Current Biology 7, 337-343.

Eischen CM, Dick CJ, Leibson PJ. 1994. Tyrosine kinase activation provides an early and requisite signal for Fas-induced apoptosis. The Journal of Immunology 153, 1947-1954.

Gibson S, Widmann C, Johnson GL. 1999. Differential involvement of MEK kinase 1 (MEKK 1) in the induction of apoptosis in response to microtubule-targeted drugs versus DNA damaging agents. The Journal of Biological Chemistry. 274, 10916-10922.

Glinsky GV, Glinsky VV, Ivanova AB, Hueser CJ. 1997. Apoptosis and metastasis: increased apoptosis resistance of metastatic cancer cells is associated with the profound deficiency of apoptosis execution mechanisms. Cancer Letters 115, 185-193.

Gunawardena AHLAN, Pearce DM, Jackson MB, Hawes CR, Evans DE. 2001.Characterisation of programmed cell death during aerenchyma formation induced by ethylene or hypoxia in roots of maize (Zea mays L.). Planta 212, 205-214.

Hagar H, Ueda N, Shah SV. 1997. Tyrosine phosphorylation in DNA damage and cell death in hypoxic injury to LLC-PK1 cells. Kidney International 51, 1747-1753.

Harris GC, Gibbs PB, Ludwig G, Un A, Sprengnether M. Kolodny N. 1986. Mannose metabolism in corn and its impact on leaf metabolites, photosynthetic gas exchange, and chlorophyll fluorescence. The Plant Physiology 82, 1081-1089.

Herold A, Lewis DH. 1977. Mannose and green plants: occurrence, physiology and metabolism, and use as tool to study the role of orthophosphate. The New Phytologist 79, 1-40.

Houot V, Etienne P, Petitot AS, Barbier S, Blein JP, Suty L. 2001. Hydrogen peroxide induces programmed cell death features in cultured tobacco BY-2 cells, in a dose-dependent manner. Journal of Experimental Botany 52, 1721-1730.

Huang H-J, Fu S-F, Tai Y-H, Chou W-C, Huang D-D. 2002. Expression of Oryzae sativa MAP Kinase Gene is Developmentally Regulated and Stress-Responsive. Physiological Plantarum. 104(4) 572-580.

Hunter T. 1987. A thousand and one protein kinases. Cell 50,823-829.

Hunter T. 1995. Protein kinases and phosphatases: The Yin and Yang of protein phosphorylation and signaling. Cell 80, 225-236.

Hunter T, Karin M. 1992. The regulation of transcription by phosphorylation. Cell 70, 375-387.

Jan MS, Wing LY, Lin MT, Lin YS. 1999. α-Difluoromethylornithine blocks thymocyte apoptosis via a reduction in tyrosine phosphorylation. Scandinavian Journal of Immunology 50, 605-611.

Jonak C, Heberle-Bors E, Hirt H. 1994. MAP kinases: universal multi-purpose signaling tools. Plant Molecular Biology 24, 407-416.

Jones AM. 2001. Programmed cell death in development and defense. Plant Physiology 125, 94-97.

Jong AJD, Hoeberichts FA, Yakimova ET, Maximova E, Woltering EJ. 2000. Chemical-induced apoptotic cell death in tomato cells: involvement of caspase-like proteases. Planta 211, 656-662.

Katsuhara M. 1997. Apoptosis-like cell death in barley roots under salt stress. Plant and Cell Physiology 38, 1091-1093.

Kawai M, Uchimiya H. 2000. Coleoptile senescence in rice (Oryza sativa L.). Annals of Botany 86, 405-414.

Kerr JF, Wyllie AH, Currie AR. 1972. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer 26, 239-257.

Kikuchi M, Tenneti L, Lipton SA. 2000. Role of p38 mitogen-activated protein kinase in axotomy-induced apoptosis of rat retinal ganglion cells. The Journal of Neuroscience 20, 5037-5044.

Kitta K, Day RM, Ikeda T, Suzuki YJ. 2001. Hepatocyte growth factor protects cardiac myocytes against oxidative stress-induced apoptosis. Free Radical Biology and Medicine 31, 902-910.

Koukalova B, Kovařik A, Fajkus J, Široky J. 1997. Chromatin fragmentation associated with apoptotic changes in tobacco cells exposed to cold stress. FEBS Letters 414, 289-292.

Kyprianou N, Bains A, Rhee JG. 1997. Transient tyrosine phosphorylation of p34cdc2 is an early event in radiation-induced apoptosis of prostate cancer cells. The Prostate 32, 266-271.

Kyriakis JM, Avruch J. 1996. Protein kinase cascades activated by stress and inflammatory cytokinases. Bioessay 18, 567-577.

Lam E, Pontier D, Pozo OD. 1999. Die and let live-programmed cell death in plants. Current Opinion in Plant Biology 2, 502-507.

Liu Y, Bhalla K, Hill C, Priest DG.1994.Evidence for involvement of tyrosine phosphorylation in taxol-induced apoptosis in a human ovarian tumor cell line. Biochemical Pharmacology 48,1265-1272.

Malca I, Endo RM, Long MR.1967. Mechanism of glucose counteraction of inhibition of root elongation by galactose, mannose, and glucosamine. Phytopathology 57, 272-278.

Marushige K, Marushige Y. 1999. Changes in the mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt signaling associated with the induction of apoptosis. Anticancer Research 19, 3865-3872.

Matheson NK, Myers DK. 1998. Inhibition of germination by glucose analogues that are hexokinase substrates. Phytochemistry 48, 241-248.

McCabe PF, Leaver CJ. 2000. Programmed cell death in cell cultures. Plant Molecular Biology 44, 359-368.

McCabe PF, Levine A, Meijer PJ, Tapon NA, Pennell RI. 1997. A programmed cell death pathway activated in carrot cells cultured at low cell density. The Plant Journal 12, 267-280.

Mittler R, Feng X, Cohen M. 1998. Post-transcriptional suppression of cytosolic ascorbate peroxidase expression during pathogen-induced programmed cell death in tobacco. The Plant Cell 10, 461-473.

Mittler R, Lam E. 1995a. In-situ detection of nDNA fragmentation during the differentiation of tracheary elements in higher plants. The Plant Physiology 108, 489-493.

Mittler R, Lam E. 1995b. Identification, characterization, and purification of a tobacco endonuclease activity induced upon hypersensitive response cell death. The Plant Cell 7, 1951-1962.

Mittler R, Shulaev V, Lam E. 1995. Coordinated activation of programmed cell death and defense mechanisms in transgenic tobacco plants expressing a bacterial proton pump. The Plant Cell 7, 29-42.

Mittler R, Shulaev V, Seskar M, Lam E. 1996. Inhibition of programmed cell death in tobacco plants during a pathogen-induced hypersensitive response at low oxygen pressure. The Plant Cell 8, 1991-2001.

Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiological Plantarum 15, 473-497.

Navarre DA, Wolpert TJ. 1999 Victorin induction of an apoptotic/senescence-like response in oats. The Plant Cell 11, 237-249.

O’Brien IEW, Murray BG, Baguley BC, Morris BAM, Ferguson IB. 1998. Major changes in oats. The Plant Cell 11, 237-249.O’Brien IEW, Murray BG, Baguley BC, Morris BAM, Ferguson IB. 1998. Major changes in chromatin condensation suggest the presence of an apoptotic pathway in plant cells. Experimental Cell Research 241, 46-54.

Orzaez D, Granell A. 1997. DNA fragmentation is regulated by ethylene during arpel senescence in Pisum sativum. The Plant Journal 11, 137-144.

Otani H, Erdos M, Leonard WJ. 1993. Tyrosine kinase(s) regulate apoptosis and bcl-2 expression in a growth factor-dependent cell line. The Journal of Biological Chemistry 268, 22733-22736.

Pan G, Zhou T, Radding W, Saag MS, Mountz JD, McDonald JM. 1998. Calmodulin antagonists inhibit apoptosis of CD4+ T-cells from patients with AIDS. Immunopharmacology 40, 91-103.

Pego JV, Weisbeek PJ, Smeekens SCM. 1999. Mannose inhibits Arabidopsis germination via a hexokinase-mediated step. The Plant Physiology 119, 1017-1023.

Pennell RI, Lamb C. 1997. Programmed cell death in plants. The Plant Cell 9,1157-1168.

Peters W, Ritter J, Tiller H, Valdes O, Renner U, Fountain M, Beck E. 2000. Growth, ageing and death of a photoautotrophic plant cell culture. Planta 210, 478-487.

Reed JC.1997. Cytochrome c: can’t live with it─can’t live without it. Cell 91, 559- 562.

Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular Cloning: A Laboratory Manual, Ed 2. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Samuel MA, Miles GP, Ellis BE. 2000. Ozone treatment rapidly activates MAP kinase signaling in plants. The Plant Journal 22, 367-376.

Saraste A, Pulkki K. 2000. Morphologic and biochemical hallmarks of apoptosis Cardiovascular Research 45, 528-537.

Scheving LA, Thomas JR, Zhang L. 1999. Regulation of intestinal tyrosine phosphorylation and programmed cell death by peroxovanadate. American Journal of Physiology 46, C572-C579.
Schulz JB, Gerhardt E. 2001. Apoptosis: its relevance to Parkinson’s disease. Clinical Neuroscience Research 1, 427-433.

Schwartz LM, Smith SW, Jones MEE, Osborne BA. 1992. Do all programmed cell deaths occur via apoptosis? Proceedings of the National Academy of Sciences of the United States of America 90, 980-984.

Seger R, Krebs EG.1995. The MAPK signaling cascade. The FASEB Journal 9,726-735.

Shimizu S, Narita M, Tsujimoto Y. 1999. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature. 399, 483-487.

Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, Wang HG, Reed JC, Nicholson DW, Alnemri ES, Green DR, Martin SJ. 1999. Ordering the cytochrome c-intiated caspase cascade: Hierarchical activation of caspases-2, -3, -6, -7, -8 and –10 in a caspase-9-dependent manner. The Journal of Cell Biology 144, 281-292.

Stein JC, Hansen G. 1999. Mannose induces an endonuclease responsible for DNA laddering in plant cells. The Plant Physiology 121, 71-79.

Stone JM, Walker JC. 1995. Plant protein kinase families and signal transduction. The Plant Physiology 108, 451-457.

Sun YL, Zhao Y, Hong X, Zhai ZH. 1999. Cytochrome c release and caspase activation during menadione-induced apoptosis in plants. FEBS Letters 462, 317-321.

Tada Y, Hata S, Takata Y, Nakayashiki H, Tosa Y, Mayama S. 2001. Induction and signaling of an apoptotic response typified by DNA laddering in the defense response of Oats to infection and elicitors. Molecular Plant-Microbe Interactions 14, 477-486.

Thompson CB. 1995. Apoptosis in the pathogenesis and treatment of disease. Science 267, 1456-1462.

Trojanek J, Ek P. Scoble, Muszynska G, Engstrom L. 1996. Phosphorylation of plant proteins and the identification of protein-tyrosine kinase activity in maize seedlings. European Journal of Biochemistry 235, 338-344.
Vaux DL, Korsmeyer SJ.1999. Cell death in development. Cell 96, 245-254.

Wang H, Li J, Bostock RM, Gilchrist DG. 1996. Apoptosis: a functional paradigm for programmed plant cell death induced by a host-selective phytotoxin and invoked during development. The Plant Cell 8, 375-391.

Wang M, Oppedijk BJ, Caspers MPM, Lamers GEM, Boot MJ, Geerlings DNG, Bakhuizen B, Meijer AH, Duijn BV. 1998. Spatial and temporal regulation of DNA fragmentation in the aleurone of germinating barley. Journal of Experimental Botany 49, 1293-1301.

Wang X, Martindale JL, Holbrook NJ. 2000. Requirement for ERK activation in cisplatin-induced apoptosis. The Journal of Biological Chemistry. 275, 39435-39443.

Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. 1995. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270, 1326-1331.

Yamada T, Marubashi W, Niwa M. 2000. Apoptotic cell death induces temperature-sensitive lethality in hybrid seedlings and calli derived from the cross of Nicotiana suaveolens × N. tabacum. Planta 211, 614-622.

Yen CH, Yang CH. 1998. Evidence for programmed cell death during leaf senescence in plants. Plant and Cell Physiology 39, 922-927.

Yousefi S, Green DR, Blaser K, Simon HU. 1994. Protein-tyrosine phosphorylation regulates apoptosis in human eosinophils and neutrophils. Proceedings of the National Academy of Sciences of the United States of America 91, 10868-10872.

Zhang S, Liu Y, Klessig DF. 2000. Multiple levels of tobacco WIPK activation during the induction of cell death by fungal elicitors. The Plant Journal 23, 339-347.

Zhao Y, Jiang ZF, Sun YL, Zhai ZH. 1999. Apoptosis of mouse liver nuclei induced in the cytosol of carrot cells. FEBS Letters 448, 197-200.

Zou H, Henzel WJ, Liu X. 1997. Apaf-1, a human protein homologous to C.elegans CED-4 participates in cytochrome c-dependent activation of caspase-3. Cell 90, 405-413.